Protein and DNA thereof

ABSTRACT

The protein of the present invention and a DNA encoding the same can be used as therapeutic/prophylactic agents for diseases such as infectious diseases. The protein of the present invention is also useful as a reagent for screening a compound or its salt capable of promoting or inhibiting the activity of the protein of the present invention. Furthermore, a compound or its salt inhibiting the activity of the protein of the present invention and a neutralization antibody inhibiting the activity of the protein of the present invention can also be used as therapeutic/prophylactic agents for diseases such as bronchial asthma, chronic obstructive pulmonary disease, etc.

This application is the National Phase filing of International PatentApplication No. PCT/JP 00/08015, filed Nov. 14, 2000.

FIELD OF THE INVENTION

This invention relates to a novel protein and its DNA, which is usefulas a diagnostic marker or a drug target for bronchial asthma, chronicobstructive pulmonary disease, etc., and also as a therapeutic agent, aprophylactic agent, or the like, for infectious diseases, immunedeficiency, etc.

BACKGROUND ART

Bronchial asthma is a chronic inflammatory disease of airways showingrespiratory stenosis, in which symptoms such as paroxysmal dyspnea,stridor, cough, etc. are observed. Many cells such as bronchialepithelial cells, mast cells, eosinophils, T lymphocytes, etc. areinvolved in its onset and development. One of the most importantcharacteristics of bronchial asthma is that airways are hyperresponsiveto irritants (airway hyperresponsiveness). This airwayhyperresponsiveness is attributable to airway inflammation caused mainlydue to exfoliation of bronchial epithelial cells by neurotransmitterssecreted from the cells such as eosinophils, etc., infiltrated intoairways, and it is also considered that genetic factors or environmentalfactors will affect the airway hyperresponsiveness complicatedly.

When the inflammatory reaction of airways is triggered by externalirritants (allergens, exhausts) or viral infection, adhesion moleculesincluding VCAM-1, ICAM-1 and the like are expressed on bronchialepithelial cells or on capillary endothelial cells around the bronchi[J. Allergy Clin. Immunol., 96, 941 (1995)] to produce cytokines orchemotactic substances. In patients with bronchial asthma, the functionof Th2 type helper T cells is activated to increase the production ofTh2 type cytokines such as IL-3, IL-4, IL-5, IL-13, GM-CSF, etc., orchemokines such as eotaxin, RANTES, etc. IL-4 or IL-13 has an activityof inducing IgE production, and IL-3 or IL-4 has an activity of inducingthe growth of mast cells. Furthermore, eosinophils differentiate andproliferate in response to IL-5, GM-CSF, etc. and infiltrate into theairways in response to eotaxin or RANTES [Allergy Asthma Proc., 20, 141(1999)].

Epithelial cells that cover the bronchial mucosa not only have thebarrier function to prevent direct transmittance of external irritantsto submucosal tissues and the function to excrete mucus secretions orforeign matters, but also regulate bronchoconstriction by secretedepithelium-derived smooth muscle relaxing factors, etc. Eosinophilsinfiltrated into the airways of patient with bronchial asthma releasethrough degranulation of intracellular granule proteins such asactivated MBP (major basic protein), ECP (eosinophil cationic protein),etc. [Compr. Ther., 20, 651 (1994)]. By the cytotoxic action of thesegranular proteins, the exfoliation and damages of epithelial cellsoccur. The exfoliation of epithelial cells leads to exposure of sensorynerve terminals, increase in epithelial permeability and loss ofepithelium-derived smooth muscle relaxing factors. Also, leukotriene C4(LTC4) or platelet activating factor (PAF) produced by eosinophilsincrease tension of bronchial smooth muscle. It is considered that whenthe foregoing changes are repeated to make it chronic, the bronchialwalls would be thickened to cause airway hyperresponsiveness.

As stated above, it is known that genes of the cytokines or adhesionmolecules described above are increasingly expressed, accompanied byinflammation of the airways, but there is no report to systematicallyanalyze the change of genes, expression of which are localized in thelesion of lung/bronchi and which are associated with the onset of airwayhyperresponsiveness.

On the other hand, chitinase activity was detected in blood plasma frompatient with Gaucher's disease [J. Clin. Invest., 93, 1288 (1994)), theprotein was purified as only one chitinase in mammal [J. Biol. Chem.,270, 2198 (1995)] and the gene was cloned [J. Biol. Chem., 270, 26252(1995)]. This chitinase has been used as a disease marker but norelationship between bronchial asthma and chitinase has not beenreported.

The present invention provides a novel protein, expression of whichincreases in the lung/bronchi having increased airwayhyperresponsiveness, or salts thereof, its partial peptide or saltsthereof, its signal peptide; a DNA encoding the protein, its partialpeptide or signal peptide; recombinant vectors; transformants; methodsof manufacturing the protein; pharmaceutical compositions comprising theprotein or DNA; antibodies against the protein; methods for screeningcompounds that suppress or promote the expression of the protein;methods for screening compounds that suppress or promote the activity ofthe protein; compounds obtainable by the screening methods; etc.

DISCLOSURE OF THE INVENTION

The present inventors made extensive studies to solve the foregoingproblems and as a result, discovered a gene, expression of whichmarkedly increases in the lung/bronchi of mouse asthma model.Furthermore based on the base sequence of this gene, the inventorssucceeded in cloning a cDNA having a novel base sequence from humangastric cDNA library, and found that a protein encoded by the cDNAbelongs to the chitinase family.

Based on these findings, the inventors continued further investigationsto accomplish the present invention.

That is, the present invention relates to the following features.

(1) A protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:1, or asalt thereof.

(2) A partial peptide of the protein according to (1), or a saltthereof.

(3) A signal peptide having the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:2, ora salt thereof.

(4) A DNA containing a DNA encoding the protein according to (1) or thepartial peptide according to (2).

(5) The DNA according to (4) having a base sequence represented by SEQID NO:3.

(6) A DNA containing a DNA encoding the signal peptide according to (3).

(7) The DNA according to (6) having a base sequence represented by SEQID NO:4.

(8) A recombinant vector containing the DNA according to (4).

(9) A transformant transformed with the recombinant vector according to(8).

(10) A method of manufacturing the protein according to (1) or thepartial peptide according to (2), or a salt thereof, which comprisesculturing said transformant according to (9), producing/accumulating theprotein according to (1) or the partial peptide according to (2), andcollecting the same.

(11) A pharmaceutical comprising the protein according to (1) or thepartial peptide according to (2) or a salt thereof.

(12) A pharmaceutical comprising the DNA according to (4).

(13) An antibody against the protein according to (1) or the partialpeptide according to (2), or a salt thereof.

(14) A method of screening a compound or a salt thereof capable ofpromoting or inhibiting the activity of the protein according to (1) orthe partial peptide according to (2), or a salt thereof, which comprisesusing the protein according to (1) or the partial peptide according to(2), or a salt thereof.

(15) A kit for screening a compound or a salt thereof capable ofpromoting or inhibiting the activity of the protein according to (1) orthe partial peptide according to (2), or a salt thereof, comprising theprotein according to (1) or the partial peptide according to (2), or asalt thereof.

(16) A compound or a salt thereof capable of promoting or inhibiting theactivity of the protein according to (1) or the partial peptideaccording to (2), or a salt thereof, which is obtainable using thescreening method according to (14) or the screening kit according to(15).

(17) A pharmaceutical comprising a compound or a salt thereof capable ofpromoting or inhibiting the activity of the protein according to (1) orthe partial peptide according to (2), or a salt thereof, which isobtainable using the screening method according to (14) or the screeningkit according to (15).

(18) A method of screening a compound or a salt thereof capable ofinhibiting the activity of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:18, its partial peptide, or a salt thereof,which comprises using the protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:18, its partial peptide, or a salt thereof.

(19) A kit for screening a compound or a salt thereof capable ofinhibiting the activity of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:18, its partial peptide, or a salt thereof,comprising the protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:18, its partial peptide, or a salt thereof.

(20) A compound or a salt thereof capable of inhibiting the activity ofa protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:18, itspartial peptide, or a salt thereof, which is obtainable using thescreening method according to (18) or the screening kit according to(19).

(21) A pharmaceutical comprising the compound or a salt thereof,according to (20).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows tissue distribution of ECF-L gene (mRNA) in normal mouseand model mouse with increased airway hyperresponsiveness, in which Lu,H, Li, Ki, Br, Thy, Sp, SI, LI, St and PBL designate lung, heart, liver,kidney, brain, thymus, spleen, small intestine, large intestine, stomachand peripheral blood lymphocyte, respectively in EXAMPLE 2.

FIG. 2 shows administration schedule in the experiment described inEXAMPLE 3.

FIG. 3 shows change in airway reactivity with time course, byadministration of acetylcholine in a dose of 500 μg/kg, wherein Ach isacetylcholine.

FIG. 4 shows change with time course in number of infiltrated cells inthe alveolar lavage fluid shown in EXAMPLE 3, wherein Mφ, Eos, Neu andLym designate macrophage, eosinophil, neutrophil and lymphocyte,respectively.

FIG. 5 shows change of ECF-L gene (mRNA) with time course in model mousewith increased airway hyperresponsiveness shown in EXAMPLE 3.

FIG. 6 indicates the expression site of ECF-L gene on frozen sectionsfrom model mouse with increased airway hyperresponsiveness and normalmouse, shown in EXAMPLE 4.

FIG. 7 shows comparison in base sequence between DNA (human ECF-L)encoding human-derived ECF-L like protein and mouse ECF-L gene (mouseECF-L).

FIG. 8 shows comparison in amino acid sequence between human-derivedECF-L like protein (human ECF-L) and mouse ECF-L gene (mouse ECF-L).

FIG. 9 shows comparison in amino acid sequence between human-derivedECF-L like protein (human ECF-L) and other proteins (humanchitotriosidase, human HC-gp39pit, human YKL-39) belonging to thechitinase family (continued to FIG. 10).

FIG. 10 shows comparison in amino acid sequence between human-derivedECF-L like protein (human ECF-L) and other proteins (humanchitotriosidase, human HC-gp39prt, human YXL-39) belonging to thechitinase family (continued from FIG. 9).

FIG. 11 shows tissue distribution of the gene (mRNA) encodinghuman-derived ECF-L like protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein of the present invention containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:1 (hereinafter sometimes referred to as proteinI of the invention) or the protein used in the present inventioncontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:18 (hereinafter sometimesreferred to as protein II) may be any protein derived from any cells ofhuman and other warm-blooded animals (e.g., guineapig, rat, mouse,chicken, rabbit, swine, sheep, bovine, monkey, etc.) such as hepatocyte,splenocyte, nerve cells, glial cells, β cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, goblet cells, endothelial cells, smooth musclecells, fibroblasts,fibrocytes, myocytes, fatcells, immune cells (e.g., macrophage, T cells,B cells, natural killer cells, mast cells, neutrophils, basophils,eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes,bone cells, osteoblasts, osteoclasts, mammary gland cells, hepatocyte orinterstitial cells; or the corresponding precursor cells, stem cells,cancer cells, etc.; or any tissues where such cells are present, such asbrain or any of brain regions (e.g., olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; the proteins mayalso be synthetic proteins.

As the amino acid sequence having substantially the same amino acidsequence as that shown by SEQ ID NO:1, there are amino acid sequenceshaving at least about 80% homology, preferably at least about 90%homology and most preferably at least about 95% homology, to the aminoacid sequence shown by SEQ ID NO:1.

Preferred examples of the protein containing substantially the sameamino acid sequence as the amino acid sequence shown by SEQ ID NO:1include proteins having substantially the same amino acid sequence asthe amino acid sequence shown by SEQ ID NO:1 and having a propertysubstantially equivalent to that of the protein having the amino acidsequence shown by SEQ ID NO:1, etc.

Examples of the properties substantially equivalent include anexpression pattern, an expression timing, chitinase activity, and thelike in the lung/bronchi. The substantially equivalent is used to meanthat the nature of these properties is equivalent qualitatively.Preferably, the expression pattern, expression timing, chitinaseactivity, etc. in the lung/bronchi are equivalent, but differences indegree such as a level of these properties, quantitative factors such asa molecular weight of the protein may be present and allowable.

Examples of the protein I of the present invention include so-calledmuteins such as proteins containing 1) the amino acid sequencerepresented by SEQ ID NO:1, of which at least 1 or 2 (preferably about 1to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are deleted; 2) the amino acid sequencerepresented by SEQ ID NO:1 , to which at least 1 or 2 (preferably about1 to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are added; 3) the amino acid sequencerepresented by SEQ ID NO:1, in which at least 1 or 2 (preferably about 1to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) aminoacids are inserted; 4) the amino acid sequencerepresented by SEQ ID NO:1, in which at least 1 or 2 (preferably about 1to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are substituted by other amino acids; or5) a combination of the above amino acid sequences.

As the amino acid sequence having substantially the same amino acidsequence as the amino acid sequence shown by SEQ ID NO:18, there areamino acid sequences having at least about 80% homology, preferably atleast about 90% homology and most preferably at least about 95%homology, to the amino acid sequence shown by SEQ ID NO:18.

Preferred examples of the protein containing substantially the sameamino acid sequence as the amino acid sequence shown by SEQ ID NO:18include proteins having substantially the same amino acid sequence asthe amino acid sequence shown by SEQ ID NO:18 and having a propertysubstantially equivalent to that of the protein having the amino acidsequence shown by SEQ ID NO:18, etc.

Examples of the properties substantially equivalent include anexpression pattern, an expression timing, and the like in thelung/bronchi. The substantially equivalent is used to mean that thenature of these properties is equivalent qualitatively. Preferably, theexpression pattern, expression timing, etc. in the lung/bronchi areequivalent, but differences in degree such as a level of theseproperties, quantitative factors such as a molecular weight of theprotein may be present and allowable.

Examples of the protein II of the present invention include so-calledmuteins such as proteins containing 1) the amino acid sequencerepresented by SEQ ID NO:18, of which at least 1 or 2 (preferably about1 to about30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are deleted; 2) the amino acid sequencerepresented by SEQ ID NO:18, to which at least 1 or 2 (preferably about1 to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are added; 3) the amino acid sequencerepresented by SEQ ID NO:18, in which at least 1 or 2 (preferably about1 to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are inserted; 4) the amino acid sequencerepresented by SEQ ID NO:18, in which at least 1 or 2 (preferably about1 to about 3, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are substituted by other amino acids; or5) a combination of the above amino acid sequences.

Throughout the specification, the proteins are represented in accordancewith the conventional way of describing proteins, that is, theN-terminus (amino terminus) at the left hand and the C-terminus(carboxyl terminus) at the right hand. In the protein I or II of thepresent invention including the protein containing the amino acidsequence shown by SEQ ID NO:1 or SEQ ID NO:18, the C-terminus is usuallyin the form of a carboxyl group (—COOH) or a carboxylate (—COO⁻) but maybe in the form of an amide (—CONH₂) or an ester (—COOR).

Examples of the ester group shown by R include a C₁₋₆ alkyl group suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈ cycloalkylgroup such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ aryl group such asphenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl such as a phenyl-C₁₋₂ alkylgroup, e.g., benzyl, phenethyl, etc.; an α-naphthyl-C₁₋₂ alkyl groupsuch as α-naphthylmethyl, etc.; pivaloyloxymethyl and the like.

Where the protein I or protein II of the present invention contains acarboxyl group (or a carboxylate) at a position other than theC-terminus, it may be amidated or esterified and such an amide or esteris also included within the protein I or II of the present invention.The ester group may be the same group as that described with respect tothe above C-terminal.

Furthermore, examples of the protein I of the present invention includevariants of the above proteins, wherein the amino group at theN-terminus (e.g., methionine residue) of the protein is protected with aprotecting group (e.g., a C₁₋₆ acyl group such as a C₁₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.); those wherein the N-terminalregion is cleaved in vivo and the glutamyl group thus formed ispyroglutaminated; those wherein a substituent (e.g., —OH, —SH, aminogroup, imidazole group, indole group, guanidino group, etc.) on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group (e.g., a C₁₋₆ acyl group such as a C₁₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.), or conjugated proteins such asglycoproteins having sugar chains.

Specific examples of the protein I of the present invention include ahuman stomach-derived protein containing the amino acid sequencerepresented by SEQ ID NO:1, and the like.

Specific examples of the protein II of the present invention include amouse-derived protein containing the amino acid sequence represented bySEQ ID NO:18, and the like.

The protein of the present invention containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:1 further includes, for example, precursorproteins, in which at least one or two amino acids, preferably about 1to about 200, more preferably about 1 to about 100, and most preferablyabout 1 to about 50, amino acids are conjugated at the N-terminus or(and) at the C-terminus of the protein I of the invention describedabove (hereinafter sometimes merely referred to as the precursor proteinI of the invention).

The precursor protein I of the invention may be proteins derived fromcells of, e.g., human and other warm-blooded animals (e.g., guineapig,rat, mouse, chicken, rabbit, swine, sheep, bovine, monkey, etc.) or anytissue in which these cells are present; or may also be syntheticproteins.

The precursor protein I of the invention may be any protein capable ofproducing the protein I of the invention described above. Thus,differences in quantitative factors such as a molecular weight of theprotein may be present and allowable.

In the precursor protein I of the invention, the C-terminus is usuallyin the form of a carboxyl group (—COOH) or a carboxylate (—COO⁻) but maybe in the form of an amide (—CONH₂) or an ester (—COOR) as in theprotein I of the invention described above.

When the precursor protein I of the present invention contains carboxylgroups (or carboxylates) other than at the C-terminus, the precursorprotein I further includes proteins wherein the carboxyl groups areamidated or esterified, those wherein the amino group of the N-terminalamino acid residue (e.g., methionine residue) is protected with aprotecting group, those wherein the N-terminal region is cleaved in vivoand the glutamine thus formed is pyroglutaminated, those wherein asubstituent on the side chain of an amino acid in the molecule isprotected with a suitable protecting group, or conjugated proteins suchas so-called glycoproteins to which sugar chains are bound, etc., as inthe protein I of the present invention described above.

Specific examples of the precursor protein I of the present inventionare a protein, in which the signal peptide of the present inventioncontaining the amino acid sequence represented by SEQ ID NO:2 laterdescribed is bound at the N-terminus of the protein I of the presentinvention represented by SEQ ID NO:1 (i.e., a protein containing theamino acid sequence represented by SEQ ID NO:5), and the like.

For example, the precursor protein I of the present invention containingthe signal peptide of the present invention later described is capableof efficiently secreting the protein I of the present inventionextracellularly. The precursor protein I is also useful as anintermediate for manufacturing the protein I of the present invention.

The precursor protein I of the present invention can exhibit theactivity similar to that of the protein I of the present invention, andcan be used similarly to the protein I of the present invention.

The protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:18 furtherincludes, for example, precursor proteins, in which at least one or twoamino acids, preferably about 1 to about 200, more preferably about 1 toabout 100, and most preferably about 1 to about 50, amino acids areconjugated at the N-terminus or (and) at the C-terminus of the proteinII described above (hereinafter sometimes merely referred to as theprecursor protein II of the invention).

The precursor protein II can be proteins derived from cells of, e.g.,human and other warm-blooded animals described above (e.g., guinea pig,rat, mouse, chicken, rabbit, swine, sheep, bovine, monkey, etc.) or anytissue in which these cells are present; or may also be syntheticproteins.

The precursor protein II may be any protein capable of producing theprotein II described above. Thus, differences in quantitative factorssuch as a molecular weight of the protein may be present and allowable.

In the precursor protein II, the C-terminus is usually in the form of acarboxyl group (—COOH) or a carboxylate (—COO⁻) but may be in the formof an amide (—CONH₂) or an ester (—COOR) as in the protein II describedabove.

When the precursor protein II contains carboxyl groups (or carboxylates)other than at the C-terminus, the precursor protein II further includesproteins wherein the carboxyl groups are amidated or esterified, thosewherein the amino group of the N-terminal amino acid residue (e.g.,methionine residue) is protected with a protecting group, those whereinthe N-terminal region is cleaved in vivo and the glutamine thus formedis pyroglutaminated, those wherein a substituent on the side chain of anamino acid in the molecule is protected with a suitable protectinggroup, or conjugated proteins such as so-called glycoproteins to whichsugar chains are bound, etc., as in the protein II described above.

Specific examples of the precursor protein II are a protein, in whichthe signal peptide containing the amino acid sequence represented by SEQID NO:18 is bound at the N-terminus of the protein II represented by SEQID NO:1 (i.e., a protein containing the amino acid sequence representedby SEQ ID NO:17), and the like.

For example, the precursor protein II containing the signal peptide(1-21 amino acid residues in the amino acid sequence represented by SEQID NO:17) is capable of efficiently secreting the protein IIextracellularly. The precursor protein II is also useful as anintermediate for manufacturing the protein II.

The precursor protein II can exhibit the activity similar to that of theprotein II, and can be used similarly to the protein II.

The partial peptide (hereinafter sometimes merely referred to as thepartial peptide I of the invention) of the protein I of the presentinvention may be any peptide as long as it is a partial peptide of theprotein I of the present invention described above and preferably hasthe property equivalent to that of the protein I of the presentinvention described above. Examples of the partial peptide I includepeptides containing at least 20, preferably at least 50, more preferablyat least 70, much more preferably at least 100, and most preferably atleast 200, amino acids in the constituent amino acid sequence of theprotein I of the present invention, and the like.

The partial peptide I of the present invention may be peptidescontaining the amino acid sequence, of which at least 1 or 2 (preferablyabout 1 to about 10 and more preferably several (1 to 5)) amino acidsare deleted; peptides, to which at least 1 or 2 (preferably about 1 toabout 10 and more preferably several (1 to 5)) amino acids are added;peptides, in which at least 1 or 2 (preferably about 1 to about 10 andmore preferably several (1 to 5)) amino acids are inserted; or peptides,in which at least 1 or 2 (preferably about 1 to about 10 and morepreferably several (1 to 5)) amino acids are substituted by other aminoacids.

In the partial peptide I of the invention, the C-terminus is usually inthe form of a carboxyl group (—COOH) or a carboxylate (—COO⁻) but may bein the form of an amide (—CONH₂) or an ester (—COOR), as in the proteinI of the present invention described above.

When the partial peptide I of the invention contains carboxyl groups (orcarboxylates) other than at the C-terminus, the partial peptide Ifurther includes proteins wherein the carboxyl groups are amidated oresterified, those wherein the amino group of the N-terminal amino acidresidue (e.g., methionine residue) is protected with a protecting group,those wherein the N-terminal region is cleaved in vivo and the glutaminethus formed is pyroglutaminated, those wherein a substituent on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group, or conjugated proteins such as so-called glycoproteinsto which sugar chains are bound, etc., as in the protein I describedabove.

The partial peptide I of the invention may also be used as an antigenfor producing antibodies.

The partial peptide (hereinafter sometimes merely referred to as thepartial peptide II of the invention) of the protein II may be anypeptide as long as it is a partial peptide of the protein II describedabove and preferably has the property equivalent to that of the proteinII described above. Examples of the partial peptide II include peptidescontaining at least 20, preferably at least 50, more preferably at least70, much more preferably at least 100, and most preferably at least 200,amino acids in the constituent amino acid sequence of the protein II,and the like.

The partial peptide II may be peptides containing the amino acidsequence, of which at least 1 or 2 (preferably about 1 to about 10 andmore preferably several (1 to 5)) amino acids are deleted; peptides, towhich at least 1 or 2 (preferably about 1 to about 10 and morepreferably several (1 to 5)) amino acids are added; peptides, in whichat least 1 or 2 (preferably about 1 to about 10 and more preferablyseveral 11 to 5)) amino acids are inserted; or peptides, in which atleast 1 or 2 (preferably about 1 to about 10 and more preferably several(1 to 5)) amino acids are substituted by other amino acids.

In the partial peptide II, the C-terminus is usually in the form of acarboxyl group (—COON) or a carboxylate (—COO⁻) but may be in the formof an amide (—CONH₂) or an ester (—COOR) as in the protein II describedabove.

When the partial peptide II contains carboxyl groups (or carboxylates)other than at the C-terminus, the partial peptide II further includesproteins wherein the carboxyl groups are amidated or esterified, thosewherein the amino group of the N-terminal amino acid residue (e.g.,methionine residue) is protected with a protecting group, those whereinthe N-terminal region is cleaved in vivo and the glutamine thus formedis pyroglutaminated, those wherein a substituent on the side chain of anamino acid in the molecule is protected with a suitable protectinggroup, or conjugated proteins such as so-called glycoproteins to whichsugar chains are bound, etc., as in the protein II of the presentinvention described above.

The partial peptide II may also be used as an antigen for producingantibodies.

As the signal peptide of the present invention, there are employed thosecontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by, for example, SEQ ID NO:2, and thelike (hereinafter sometimes merely referred to as the signal peptide Iof the present invention).

The signal peptide I of the invention may be proteins derived from cellsof, e.g., human and other warm-blooded animals described above (e.g.,guinea pig, rat, mouse, chicken, rabbit, swine, sheep, bovine, monkey,etc.) or any tissue in which these cells are present; or may also besynthetic peptides.

As the amino acid sequence having substantially the same amino acidsequence as the amino acid sequence shown by SEQ ID NO:2, there areamino acid sequences having at least about 70% homology, preferably atleast about 80% homology, more preferably at least about 90% and mostpreferably at least about 95% homology, to the amino acid sequence shownby SEQ ID NO:2. More specifically, the signal peptide may be any peptidehaving substantially the same amino acid sequence as the amino acidsequence shown by SEQ ID NO:2 and capable of exhibiting the function asa signal peptide. Thus, differences in quantitative factors such as amolecular weight of the protein may be present and allowable.

The signal peptide I of the present invention may be peptides containingthe amino acid sequence, of which at least 1 or 2 (preferably about 1 toabout 10 and more preferably several (1 to 5)) amino acids are deleted;peptides, to which at least 1 or 2 (preferably about 1 to about 10 andmore preferably several (1 to 5)) amino acids are added; peptides, inwhich at least 1 or 2 (preferably about 1 to about 10 and morepreferably several (1 to 5)) amino acids are inserted; or peptides, inwhich at least 1 or 2 (preferably about 1 to about 10 and morepreferably several (1 to 5)) amino acids are substituted by other aminoacids.

In the signal peptide I of the invention, the C-terminus is usually inthe form of a carboxyl group (—COOH) or a carboxylate (—COO⁻) but may bein the form of an amide (—CONH₂) or an ester (—COOR), as in the proteinI of the present invention described above.

When the signal peptide I of the invention contains carboxyl groups (orcarboxylates) other than at the C-terminus, the signal peptide I furtherincludes proteins wherein the carboxyl groups are amidated oresterified, those wherein the amino group of the N-terminal amino acidresidue (e.g., methionine residue) is protected with a protecting group,those wherein the N-terminal region is cleaved in vivo and the glutaminethus formed is pyroglutaminated, those wherein a substituent on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group, or conjugated proteins such as so-called glycoproteinsto which sugar chains are bound, etc., as in the protein I of thepresent invention described above.

Specific examples of the signal peptide I of the present invention arepeptides containing the amino acid sequence represented by SEQ ID NO:2,in which the protein I of the present invention containing the aminoacid sequence represented by SEQ ID NO:1 has been removed from theprecursor protein I of the present invention containing the amino acidsequence represented by SEQ ID NO:5, and the like.

The signal peptide I of the present invention is capable of efficientlysecreting a variety of extracellular secretory proteins including theprotein I of the present invention extracellularly.

The protein I, precursor protein I, partial peptide I or signal peptideI, or protein II, precursor protein II, partial peptide II or signalpeptide II, of the present invention may be used in the form of saltswith physiologically acceptable acids (e.g., inorganic acids or organicacids) or bases (e.g., alkali metal salts), preferably in the form ofphysiologically acceptable acid addition salts. Examples of such saltsare salts with inorganic acids (e.g., hydrochloric acid, phosphoricacid, hydrobromic acid, sulfuric acid), salts with organic acids (e.g.,acetic acid, formic acid, propionic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The protein I, precursor protein I, partial peptide I or signal peptideI, or protein II, precursor protein II, partial peptide II or signalpeptide II, of the present invention or salts thereof maybe manufacturedby a publicly known method used to purify a protein from human or otherwarm-blooded animal cells or tissues described above. Alternatively,they may also be manufactured by culturing transformants containing DNAsencoding these proteins or peptides. Furthermore, they may also bemanufactured by a modification of the methods for peptide synthesis,which will be described hereinafter.

Where these proteins or peptides are manufactured from human ormammalian tissues or cells, human or mammalian tissues or cells arehomogenized, then extracted with an acid or the like, and the extract isisolated and purified by a combination of chromatography techniques suchas reverse phase chromatography, ion exchange chromatography, and thelike.

To synthesize the protein I, precursor protein I, partial peptide I orsignal peptide I, or protein II, precursor protein II, partial peptideII or signal peptide II, of the present invention, or amides thereof,commercially available resins that are used for protein synthesis may beused. Examples of such resins include chloromethyl resin, hydroxymethylresin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzylalcohol resin, 4-methylbenzhydrylamine resin, PAM resin,4-hydroxymethylmethylphenyl acetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin, etc. Using theseresins, amino acids in which α-amino groups and functional groups on theside chains are appropriately protected are condensed on the resin inthe order of the sequence of the objective protein or peptide accordingto various condensation methods publicly known in the art. At the end ofthe reaction, the protein or peptide is excised from the resin and atthe same time, the protecting groups are removed. Then, intramoleculardisulfide bond-forming reaction is performed in a highly dilutedsolution to obtain the objective protein or peptide, or amides thereof.

For condensation of the protected amino acids described above, a varietyof activation reagents for protein synthesis may be used, butcarbodiimides are particularly preferably employed. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin, or the protected amino acids are previously activated in the formof symmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

Solvents suitable for use to activate the protected amino acids orcondense with the resin may be chosen from solvents that are known to beusable for protein condensation reactions. Examples of such solvents areacid amides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxane, tetrahydrofuran, etc.; nitrites such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to proteinbinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedusing the ninhydrin reaction; when the condensation is insufficient, thecondensation can be completed by repeating the condensation reactionwithout removal of the protecting groups. When the condensation is yetinsufficient even after repeating the reaction, unreacted amino acidsare acetylated with acetic anhydride or acetylimidazole to cancel anypossible adverse affect on the subsequent reaction.

Examples of the protecting groups used to protect the starting aminogroups include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl,diphenylphosphinothioyl, Fmoc, etc.

A carboxyl group can be protected by, e.g., alkyl esterification (in theform of linear, branched or cyclic alkyl esters of the alkyl moiety suchas methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification(e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.),phenacyl esterification, benzyloxycarbonyl hydrazidation,t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

The hydroxyl group of serine can be protected through, for example, itsesterification or etherification. Examples of groups appropriately usedfor the esterification include a lower C₁₋₆ alkanoyl group, such asacetyl group, an aroyl group such as benzoyl group, and a group derivedfrom carbonic acid such as benzyloxycarbonyl group and ethoxycarbonylgroup. Examples of a group appropriately used for the etherificationinclude benzyl group, tetrahydropyranyl group, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethyl-benzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting amino acidsinclude the corresponding acid anhydrides, azides, activated esters(esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)). As the activated aminoacids in which the amino groups are activated in the starting material,the corresponding phosphoric amides are employed.

To eliminate (split off) the protecting groups, there are used catalyticreduction under hydrogen gas flow in the presence of a catalyst such asPd-black or Pd-carbon; an acid treatment with anhydrous hydrogenfluoride, methanesulfonic acid, trifluoromethanesulfonic acid ortrifluoroacetic acid, or a mixture solution of these acids; a treatmentwith a base such as diisopropylethylamine, triethylamine, piperidine orpiperazine; and reduction with sodium in liquid ammonia. The eliminationof the protecting group by the acid treatment described above is carriedout generally at a temperature of approximately −20° C. to 40° C. In theacid treatment, it is efficient to add a cation scavenger such asanisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide,1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, 2,4-dinitrophenylgroup known as the protecting group for the imidazole of histidine isremoved by a treatment with thiophenol. Formyl group used as theprotecting group of the indole of tryptophan is eliminated by theaforesaid acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by a treatment with an alkali such as adilute sodium hydroxide solution and dilute ammonia.

Protection of functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from publicly known groupsand publicly known means.

In another method for obtaining the amides of the desired protein orpeptide, for example, the α-carboxyl group of the carboxy terminal aminoacid is first protected by amidation; the peptide (protein) chain isthen extended from the amino group side to a desired length. Thereafter,a protein or peptide, in which only the protecting group of theN-terminal α-amino group of the peptide chain has been eliminated, and aprotein or peptide, in which only the protecting group of the C-terminalcarboxyl group has been eliminated are manufactured. The two proteins orpeptides are condensed in a mixture of the solvents described above. Thedetails of the condensation reaction are the same as described above.After the protected protein or peptide obtained by the condensation ispurified, all the protecting groups are eliminated by the methoddescribed above to give the desired crude protein or peptide. This crudeprotein or peptide is purified by various known purification means.Lyophilization of the major fraction gives the amide of the desiredprotein or peptide.

To prepare the esterified protein or peptide, for example, theα-carboxyl group of the carboxy terminal amino acid is condensed with adesired alcohol to prepare the amino acid ester, which is followed byprocedure similar to the preparation of the amidated protein or peptideabove to give the desired esterified protein or peptide.

The partial protein I, signal peptide I or partial peptide II, or saltthereof, of the present invention can be manufactured by publicly knownmethods for peptide synthesis, or by cleaving the protein I, precursorprotein I, protein II or precursor protein II, of the present inventionwith an appropriate peptidase. For the methods for peptide synthesis,for example, either solid phase synthesis or liquid phase synthesis maybe used. That is, the partial peptide or amino acids that can constructthe partial peptide or signal peptide of the present invention arecondensed with the remaining part. Where the product contains protectinggroups, these protecting groups are removed to give the desired peptide.Publicly known methods for condensation and elimination of theprotecting groups are described in 1) to 5) below.

1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, IntersciencePublishers, New York (1966)

2) Schroeder & Luebke: The Peptide, Academic Press, New York (1965)

3) Nobuo Izumiya, et al.: Peptide Gosel-no-Kiso to Jikken (Basics andexperiments of peptide synthesis), published by Maruzen Co. (1975)

4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza(Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry ofProteins) IV, 205 (1977)

5) Haruaki Yajima ed.: Zoku zyakuhin no Kaihatsu (A sequel toDevelopment of Pharmaceuticals), Vol. 14, Peptide Synthesis, publishedby Hirokawa Shoten

After completion of the reaction, the product may be purified andisolated by a combination of conventional purification methods such assolvent extraction, distillation, column chromatography, liquidchromatography and recrystallization to give the protein or peptide ofthe present invention. When the protein or peptide obtained by the abovemethods is in a free form, the peptide can be converted into anappropriate salt by a publicly known method; when the protein isobtained in a salt form, it can be converted into a free form or otherdifferent salt form by a publicly known method.

The DNA encoding the protein I or protein II of the present inventionmay be any DNA so long as it contains the base sequence encoding theprotein I or protein II of the present invention described above. TheDNA may also be any one of genomic DNA, genomic DNA library, cDNAderived from the cells or tissues described above, cDNA library-derivedfrom the cells or tissues described above and synthetic DNA.

The vector to be used for the library may be any of bacteriophage,plasmid, cosmid, phagemid and the like. In addition, the DNA can beamplified by reverse transcriptase polymerase chain reaction(hereinafter abbreviated as RT-PCR) with total RNA or mRNA fractionprepared from the above-described cells or tissues.

Specifically, the DNA encoding the protein I of the present inventionmay be any one of, for example, a DNA having the base sequencerepresented by SEQ ID NO:3, or any DNA having a base sequencehybridizable to a DNA having the base sequence represented by SEQ IDNO:3 under high stringent conditions and encoding a protein which hasthe properties substantially equivalent to those of the protein I of thepresent invention. The DNA encoding the protein II of the presentinvention may be any one of, for example, a DNA containing the 72-1142base sequence in the base sequence represented by SEQ ID NO:14, or anyDNA having a base sequence hybridizable to a DNA containing the 72-1142base sequence in the base sequence represented by SEQ ID NO:14 underhigh stringent conditions and encoding a protein which has theproperties substantially equivalent to those of the protein II of thepresent invention.

Specific examples of the DNA that is hybridizable to DNA having the basesequence represented by SEQ ID NO:3 under high stringent conditionsinclude DNAs having at least about 80% homology, preferably at leastabout 90% homology and most preferably at least about 95% homology, tothe base sequence represented by SEQ ID NO:3.

Specific examples of the DNA that is hybridizable to DNA containing the72-1142 base sequence in the base sequence represented by SEQ ID NO:14under high stringent conditions include DNAs having at least about 80%homology, preferably at least about 90% homology and most preferably atleast about 95% homology, to the 72-1142 base sequence in the basesequence represented by SEQ ID NO:14.

The hybridization can be carried out by publicly known methods or by amodification thereof, for example, according to the method described inMolecular Cloning, 2nd Ed., J. Sambrook et al., Cold Spring Harbor Lab.Press, (1989). A commercially available library may also be usedaccording to the instructions of the attached manufacturer's protocol.The hybridization can be carried out preferably under high stringentconditions.

The high stringent conditions used herein are, for example, those in asodium concentration at about 19 mM to about 40 mM, preferably about 19mM to about 20 mM at a temperature of about 50° C. to about 70° C.,preferably about 60° C. to about 6520 C. In particular, hybridizationconditions in a sodium concentration at about 19 mM at a temperature ofabout 65° C. are most preferred.

More specifically, as the DNA encoding the protein having the amino acidsequence represented by SEQ ID NO:1, there may be employed a DNAcontaining a DNA having the base sequence represented by SEQ ID NO:3.

As the DNA encoding the protein having the amino acid sequencerepresented by SEQ ID NO:18, more specifically, a DNA containing a DNAhaving the 72-1142 base sequence in the base sequence represented by SEQID NO:14 maybe employed.

The DNA encoding the precursor protein I or precursor protein II of thepresent invention may be any DNA so long as it contains the basesequence encoding the precursor protein I or precursor protein II of thepresent invention described above. The DNA may also be any of genomicDNA, genomic DNA library, cDNA derived from the cells and tissuesdescribed above, cDNA library derived from the cells and tissuesdescribed above and synthetic DNA.

Specifically, the DNA encoding the precursor protein I of the presentinvention may be any one of, for example, a DNA having the base sequencerepresented by SEQ ID NO:16, or any DNA having a base sequencehybridizable to a DNA having the base sequence represented by SEQ IDNO:16 and encoding a protein capable of producing the protein I of thepresent invention described above.

The DNA encoding the precursor protein II may be, for example, a DNAcontaining the 9-1142 base sequence in the base sequence represented bySEQ ID NO:14, or a DNA having a base sequence hybridizable to the 9-1142base sequence in the base sequence represented by SEQ ID NO:14 underhigh stringent conditions and encoding a protein capable of producingthe protein II.

As the DNA hybridizable to the DNA having the base sequence representedby SEQ ID NO:16 under high stringent conditions, there are, for example,DNAs containing the base sequences having at least about 80% homology,preferably at least about 90% homology and most preferably at leastabout 95% homology, to the base sequence shown by SEQ ID NO:16.

As the DNA hybridizable to the DNA having the 9-1142 base sequence inthe base sequence represented by SEQ ID NO:14under high stringentconditions, there are, for example, DNAs containing the base sequenceshaving at least about 80% homology, preferably at least about 90%homology and most preferably at least about 95% homology, to the 9-1142base sequence in the base sequence represented by SEQ ID NO:14.

Methods for the hybridization and the high stringent conditions that canbe used are the same as those described above.

More specifically, DNAs containing a DNA having the base sequencerepresented by SEQ ID NO:16, and the like, are employed as the DNAencoding the precursor protein I of the present invention containing theamino acid sequence represented by SEQ ID NO:5.

As the DNA encoding the precursor protein II containing the amino acidsequence represented by SEQ ID NO:17, more specifically, DNAS containingDNAs having the 9-1142 base sequence in the base sequence represented bySEQ ID NO:14, and the like, are employed.

The DNA encoding the partial peptide I or partial peptide II of thepresent invention may be any peptide so long as it contains a basesequence encoding the partial peptide I or partial peptide II of thepresent invention described above. The DNA may also be any one ofgenomic DNA, genomic DNA library, cDNA derived from the cells or tissuesdescribed above, cDNA library derived from the cells or tissuesdescribed above and synthetic DNA.

As the DNA encoding the partial peptide I of the present invention,there are employed, for example, a DNA having a part of the basesequence represented by SEQ ID NO:3, a DNA having a base sequencehybridizable to a DNA having the base sequence represented by SEQ IDNO:3 under high stringent conditions and containing a part of DNAencoding a protein having the properties substantially equivalent tothose of the protein I of the present invention, and the like.

The DNA hybridizable to the DNA having the base sequence represented bySEQ ID NO:3 has the same significance as described above.

As the DNA encoding the partial peptide II, there are employed, forexample, a DNA having a part of the 72-1142 base sequence in the basesequence represented by SEQ ID NO:14, a DNA having a base sequencehybridizable to a DNA having the 72-1142 base sequence in the basesequence represented by SEQ ID NO:14 under high stringent conditions andcontaining a part of DNA encoding a protein having the propertiessubstantially equivalent to those of the protein II, and the like.

The DNA hybridizable to the DNA having the 72-1142 base sequence in thebase sequence represented by SEQ ID NO:14 has the same significance asdescribed above.

Methods for the hybridization and the high stringent conditions that canbe used are the same as those described above.

The DNA encoding the signal peptide I of the present invention may beany peptide so long as it contains a base sequence encoding the signalpeptide I of the present invention described above. The DNA may also beany one of genomic DNA, genomic DNA library, cDNA derived from the cellsor tissues described above, cDNA library derived from the cells ortissues described above and synthetic DNA.

As the DNA encoding the signal peptide I of the present invention, thereare employed, for example, a DNA having the base sequence represented bySEQ ID NO:4, a DNA having a base sequence hybridizable to a DNA havingthe base sequence represented by SEQ ID NO:4 under high stringentconditions and encoding a peptide capable of exhibiting the function asa signal peptide, and the like.

As the DNA hybridizable to the DNA having the base sequence representedby SEQ ID NO:4 under high stringent conditions, there are employed, forexample, DNAs containing the base sequence having at least about 70%homology, preferably at least about 80% homology, more preferably atleast about 90% homology and most preferably at least about 95%homology, to the base sequence represented by SEQ ID NO:4.

Methods for the hybridization and the high stringent conditions that canbe used are the same as those described above.

More specifically, a DNA containing a DNA having the base sequencerepresented by SEQ ID NO:4, and the like, are employed as the DNAencoding the signal peptide I of the present invention containing theamino acid sequence represented by SEQ ID NO:2.

For cloning of DNAs that completely encode the protein I, precursorprotein I, partial peptide I, signal peptide I, protein II, precursorprotein II or partial peptide II, of the present invention (hereinaftersometimes merely referred to as the protein of the present invention),the DNA may be either amplified by publicly known PCR using syntheticDNA primers containing a part of the base sequence of the protein of thepresent invention, or the DNA inserted into an appropriate vector can bescreened by hybridization with a labeled DNA fragment or synthetic DNAthat encodes apart or entire region of the protein of the presentinvention. The hybridization can be carried out, for example, accordingto the method described in Molecular Cloning, 2nd (J. Sambrook et al.,Cold Spring Harbor Lab. Press, 1989). Where the hybridization is carriedout using commercially available library, the procedures may beconducted in accordance with the protocol described in the attachedinstructions.

Substitution of the base sequence of DNA can be effected by publiclyknown methods such as the ODA-LA PCR method, the Gapped duplex method orthe Kunkel method or its modification by using a publicly known kitavailable as Mutant™-super Express Km or Mutan™-K (both manufactured byTakara Shuzo Co., Ltd., trademark), etc.

The cloned DNA encoding the protein of the present invention can be usedas it is, depending upon purpose or, if desired, after digestion with arestriction enzyme or after addition of a linker thereto. The DNA maycontain ATG as a translation initiation codon at the 5′ end thereof andTAA, TGA or TAG as a translation termination codon at the 3′ endthereof. These translation initiation and termination codons may also beadded by using an appropriate synthetic DNA adapter.

The expression vector of the protein of the present invention can bemanufactured, for example, by (a) excising the desired DNA fragment fromthe DNA encoding the protein of the present invention, (b) and thenligating the DNA fragment with an appropriate expression vectordownstream a promoter in the vector.

Examples of the vector include plasmids derived form E. coli (e.g.,pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis(e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19,pSH15), bacteriophages such as λ phage, etc., animal viruses such asretrovirus, vaccinia virus, baculovirus, etc. as well is as pA1-11,pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

The promoter used in the present invention may be any promoter if itmatches well with a host to be used for gene expression. In the case ofusing animal cells as the host, examples of the promoter include SRαpromoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter,etc.

Among them, CMV (cytomegalovirus) promoter or SRλ promoter is preferablyused. Where the host is bacteria of the genus Escherichia, preferredexamples of the promoter include trp promoter, lac promoter, recApromoter, γPL promoter, lpp promoter, T7 promoter, etc. In the case ofusing bacteria of the genus Bacillus as the host, preferred example ofthe promoter are SPO1 promoter, SPO2 promoter, penP promoter, etc. Whenyeast is used as the host, preferred examples of the promoter are PHO5promoter, PGK promoter, GAP promoter, ADH promoter, etc. When insectcells are used as the host, preferred examples of the promoter includepolyhedrin prompter, P10 promoter, etc.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a poly A additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV40ori), etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene(hereinafter sometimes abbreviated as AMp^(r)), neomycin resistant gene(hereinafter sometimes abbreviated as Neo, G418 resistance), etc. Inparticular, when dhfr gene is used as the selection marker using dhfrgene-deficient Chinese hamster cells, selection can also be made on athymidine free medium.

If necessary, a signal sequence that matches with a host is added to theN-terminus of the protein of the present invention. Examples of thesignal sequence that can be used are PhoA signal sequence, OmpA signalsequence, etc. in the case of using bacteria of the genus Escherichia asthe host; α-amylase signal sequence, subtilisin signal sequence, etc. inthe case of using bacteria of the genus Bacillus as the host; MFα signalsequence, SUC2 signal sequence, etc. in the case of using yeast as thehost; and insulin signal sequence, α-interferon signal sequence,antibody molecule signal sequence, etc. in the case of using animalcells as the host, respectively.

Using the vector containing the DNA encoding the protein of the presentinvention thus constructed, transformants can be manufactured.

Examples of the host, which may be employed, are bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects and animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 [Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)], JM103 (Nucleic Acids Research, 9, 309 (1981)], JA221 [Journalof Molecular Biology, 120, 517 (1978)], HB101 [Journal of MolecularBiology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal ofBiochemistry, 95, 87 (1984)], etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cell (Sf cell), MG1 cell derived from mid-intestine ofTrichoplusia ni, High Five™ cell derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cell (BmN cell),etc. is used. Examples of the Sf cell which can be used are Sf9 cell(ATCC CRL1711), Sf21 cell (both cells are described in Vaughn, J. L. etal., In Vivo, 13, 213-217 (1977), etc.

As the insect, for example, a larva of Bombyx mori can be used [Maeda etal., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinesehamster cell CHO (hereinafter referred to as CHO cell), dhfrgene-deficient Chinese hamster cell CHO (hereinafter simply referred toas CHO (dhfr⁻) cell), mouse L cell, mouse AtT-20, mouse myeloma cell,rat GH 3, human FL cell, etc.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A.,69,2110 (1972), Gene, 17,107(1982), etc.

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979), etc.

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55(1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995) (published by Shujunsha), or Virology, 52,456 (1973).

Thus, the transformants transformed with the expression vectorscontaining the DNAs encoding the protein of the present invention can beobtained.

Where the host is bacteria belonging to the genus Escherichia or thegenus Bacillus, the transformant can be appropriately cultured in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, etc. Examples of the carbon sources include glucose, dextrin,soluble starch, sucrose, etc.; examples of the nitrogen sources includeinorganic or organic materials such as ammonium salts, nitrate salts,corn steep liquor, peptone, casein, meat extract, soybean cake, potatoextract, etc.; and, examples of the inorganic materials are calciumchloride, sodium dihydrogenphosphate, magnesium chloride, etc. Inaddition, yeast, vitamins, growth promoting factors etc. may also beadded to the medium. Preferably, pH of the medium is adjusted to about 5to about 8.

A preferred example of the medium for culturing the bacteria belongingto the genus Escherichia is M9 medium supplemented with glucose andCasamino acids [Miller, Journal of Experiments in Molecular Genetics,431-433, Cold Spring Harbor Laboratory, New York, 1972]. If necessary, achemical such as 3β-indolylacrylic acid can be added to the mediumthereby to activate the promoter efficiently.

Where the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultivated at about 15° C. to about43° C. for about 3 hours to about 24 hours. If necessary, the culturemaybe aerated or agitated.

Where the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultured generally at about 30° C. to about 40° C.for about 6 hours to about 24 hours. If necessary, the culture can beaerated or agitated.

Where yeast is used as the host, the transformant is cultivated, forexample, in Burkholder's minimal medium [Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in SD medium supplementedwith 0.5% Casamino acids [Bitter, G. A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)]. Preferably, pH of the medium is adjusted toabout 5 to about 8. In general, the transformant is cultivated at about20° C. to about 35° C. for about 24 hours to about 72 hours. Ifnecessary, the culture can be aerated or agitated.

Where insect cells or insects are used as the host, the transformant iscultivated in, for example, Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) to which an appropriate additive such asimmobilized 10% bovine serum is added. Preferably, pH of the medium isadjusted to about 6.2 to about 6.4. Normally, the transformant iscultivated at about 27° C. for about 3 days to about 5 days and, ifnecessary, the culture can be aerated or agitated.

Where animal cells are employed as the host, the transformant iscultured in, for example, MEM medium containing about 5% to about 20%fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], RPMI 1640 medium [The Journal of the American MedicalAssociation, 199, 519 (1967)], 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 hours to about60 hours and, if necessary, the culture can be aerated or agitated.

As described above, the protein of the present invention can be producedin the cell membrane of the transformant.

The protein of the present invention can be separated and purified fromthe culture described above by the following procedures.

When the protein of the present invention is extracted from the cultureor cells, the transformant or cell is collected after culturing by apublicly known method and suspended in a appropriate buffer. Thetransformant or cell is then disrupted by publicly known methods such asultrasonication, a treatment with lysozyme and/or freeze-thaw cycling,followed by centrifugation, filtration, etc. Thus, the crude extract ofthe protein can be obtained. The buffer used for the procedures maycontain a protein modifier such as urea or guanidine hydrochloride, or asurfactant such as Triton X-100™, etc. When the protein of the presentinvention is secreted in the culture broth, the supernatant can beseparated, after completion of the cultivation, from the transformant orcell to collect the supernatant by a publicly known method.

The supernatant or the protein of the present invention contained in theextract thus obtained can be purified by appropriately combining thepublicly known methods for separation and purification. Such publiclyknown methods for separation and purification include a method utilizingdifference in solubility such as salting out, solvent precipitation,etc.; a method mainly utilizing difference in molecular weight such asdialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gelelectrophoresis, etc.; a method utilizing difference in electric chargesuch as ion exchange chromatography, etc.; a method utilizing differencein specific affinity such as affinity chromatography, etc.; a methodutilizing difference in hydrophobicity such as reverse phase highperformance liquid chromatography, etc.; a method utilizing differencein isoelectric point such as isoelectrofocusing electrophoresis; and thelike.

When the protein of the present invention thus obtained is in a freeform, the protein can be converted into the salt by publicly knownmethods or modifications thereof. On the other hand, when the protein isobtained in the form of a salt, it can be converted into the free formor in the form of a different salt by publicly known methods ormodifications thereof.

The protein of the present invention produced by the recombinant can betreated, prior to or after the purification, with an appropriateprotein-modifying enzyme so that the protein can be appropriatelymodified to partially remove the polypeptide. Examples of theprotein-modifying enzyme include trypsin, chymotrypsin, arginylendopeptidase, protein kinase, glycosidase and the like.

The presence of the thus produced protein of the present invention canbe determined by a binding test to a labeled ligand and by an enzymeimmunoassay using a specific antibody.

The antibodies to the protein I, precursor protein I, partial peptide I,protein II, precursor protein II, partial peptide II, or its salts, ofthe present invention may be any of polyclonal and monoclonalantibodies, as long as they are capable of recognizing the protein I,precursor protein I, partial peptide I, protein II, precursor proteinII, partial peptide II, or its salts, of the present invention.

The antibodies to the protein I, precursor protein I, partial peptide I,protein II, precursor protein II, partial peptide II, or its salts, ofthe present invention (hereinafter these are sometimes referred tocollectively as the protein of the invention) may be produced by apublicly known method of producing an antibody or antiserum, using theprotein of the invention as an antigen.

[Preparation of Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-producing Cells

The protein of the present invention is administered to warm-bloodedanimals either solely or together with carriers or diluents to the sitewhere the production of antibody is possible by the administration. Inorder to potentiate the antibody productivity upon the administration,complete Freund's adjuvants or incomplete Freund's adjuvants may beadministered. The administration is usually carried out once every twoto six weeks and two to ten times in total. Examples of the applicablewarm-blooded animals are monkeys, rabbits, dogs, guinea pigs, mice,rats, sheep, goats and chickens, with the use of mice and rats beingpreferred.

In the preparation of monoclonal antibody-producing cells, awarm-blooded animal, e.g., mice, immunized with an antigen wherein theantibody titer is noted is selected, then spleen or lymph node iscollected after two to five days from the final immunization andantibody-producing cells contained therein-are fused with myeloma cellsfrom homozoic or heterozoic animal to give monoclonal antibody-producinghybridomas. Measurement of the antibody titer in antisera may be carriedout, for example, by reacting a labeled protein, which will be describedlater, with the antiserum followed by assaying the binding activity ofthe labeling agent bound to the antibody. The fusion may be carried out,for example, by the known method by Koehler and Milstein [Nature, 256,495, (1975)]. Examples of the fusion accelerator are polyethylene glycol(PEG), Sendai virus, etc., of which PEG is preferably employed.

Examples of the myeloma cells are those collected from warm-bloodedanimals such as NS-1, P3U1, SP2/0, AP-1, etc. In particular, P3U1 ispreferably employed. A preferred ratio of the count of theantibody-producing cells used (spleen cells) to the count of myelomacells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by incubation at 20 to 40° C.,preferably at 30 to 37° C. for 1 to 10 minutes, an efficient cell fusioncan be carried out.

Various methods can be used for screening of monoclonalantibody-producing hybridomas. Examples of such methods include a methodwhich comprises adding the supernatant of a hybridoma to a solid phase(e.g., a microplate) adsorbed with the protein as an antigen directly ortogether with a carrier, adding an anti-immunoglobulin antibody (wheremouse cells are used for the cell fusion, anti-mouse immunoglobulinantibody is used) labeled with a radioactive substance or an enzyme orProtein A and detecting the monoclonal antibody bound to the solidphase, and a method which comprises adding the supernatant of hybridomato a solid phase adsorbed with an anti-immunoglobulin antibody orProtein A, adding the protein labeled with a radioactive substance or anenzyme and detecting the monoclonal antibody bound to the solid phase,or the like.

The monoclonal antibody can be screened according to publicly knownmethods or their modifications. In general, the screening can beeffected in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any screening and growthmedium can be employed as far as the hybridoma can grow there. Forexample, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1 to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui seiyaku Co., Ltd.) and thelike, can be used for the screening and growth medium. The culture iscarried out generally at 20° C. to 40° C., preferably at 37° C., forabout 5 days to about 3 weeks, preferably 1 to 2 weeks, normally in 5%CO₂. The antibody titer of the culture supernatant of a hybridoma can bedetermined as in the assay for the antibody titer in antisera describedabove.

(b) Purification of Monoclonal Antibody

Separation and purification of a monoclonal antibody can be carried outby publicly known methods, such as separation and purification ofimmunoglobulins [for example, salting-out, alcohol precipitation,isoelectric point precipitation, electrophoresis, adsorption anddesorption with ion exchangers (e.g., DEAE), ultracentrifugation, gelfiltration, or a specific purification method which comprises collectingonly an antibody with an activated adsorbent such as an antigen-bindingsolid phase, Protein A or Protein G and dissociating the binding toobtain the antibody.]

[Preparation of Polyclonal Antibody]

The polyclonal antibody of the present invention can be manufactured bypublicly known methods or modifications thereof. For example, awarm-blooded animal is immunized with an immunogen (protein antigen) perse, or a complex of immunogen and a carrier protein is formed and awarm-blooded animal is immunized with the complex in a manner similar tothe method described above for the manufacture of monoclonal antibodies.The product containing the antibody to the protein of the presentinvention is collected from the immunized animal followed by separationand purification of the antibody.

In the complex of immunogen and carrier protein used to immunize awarm-blooded animal, the type of carrier protein and the mixing ratio ofcarrier to hapten may be any type and in any ratio, as long as theantibody is efficiently produced to the hapten immunized by crosslinkingto the carrier. For example, bovine serum albumin, bovine thyroglobulinor hemocyanin is coupled to hapten in a carrier-to-hapten weight ratioof approximately 0.1 to 20, preferably about 1 to about 5.

A variety of condensation agents can be used for the coupling of carrierto hapten. Glutaraldehyde, carbodiimide, maleimide activated ester andactivated ester reagents containing thiol group or dithiopyridyl groupare used for the coupling.

The condensation product is administered to warm-blooded animals eithersolely or together with carriers or diluents to the site that canproduce the antibody by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once every 2 to 6 weeks and 3 to 10 timesin total.

The polyclonal antibody can be collected from the blood, ascites, etc.,preferably from the blood of warm-blooded animal immunized by the methoddescribed above.

The polyclonal antibody titer in antiserum can be assayed by the sameprocedure as that for the determination of serum antibody titerdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for the separation andpurification of immunoglobulins performed as in the separation andpurification of monoclonal antibodies described hereinabove.

The antisense DNA having a complementary or substantial complementarybase sequence to the DNA encoding the protein I, precursor protein I,partial peptide I, signal peptide I, protein II, precursor protein II orpartial peptide II, of the present invention (hereinafter these DNAs aresometimes collectively referred to as the DNA of the present inventionin the following description of antisense DNA) can be any antisense DNA,so long as it possesses a base sequence complementary or substantiallycomplementary to that of the DNA of the present invention and capable ofsuppressing expression of the DNA.

The base sequence substantially complementary to the DNA of the presentinvention may, for example, be a base sequence having at least about 70%homology, preferably at least about 80% homology, more preferably atleast about 90% homology and most preferably at least about95% homology,to the full-length base sequence or partial base sequence of the basesequence complementary to the DNA of the present invention (i.e.,complementary strand to the DNA of the present invention), and the like.In the entire base sequence of the complementary strand to the DNA ofthe present invention, an antisense DNA having at least about 70%homology, preferably at least about 80% homology, more preferably atleast about 90% homology and most preferably at least about 95%homology, to the complementary strand of the base sequence which encodesthe N-terminal region of the protein of the present invention (e.g., thebase sequence around the initiation codon). These antisense DNAs can besynthesized using a publicly known DNA synthesizer, etc.

Hereinafter, the protein I, precursor protein I or partial peptide I, orits salts, of the present invention (hereinafter sometimes merelyreferred to as the protein a of the present invention), the protein II,precursor protein II or partial peptide II, or salts thereof(hereinafter sometimes merely referred to as the protein b of thepresent invention), the DNA encoding the protein a (hereinaftersometimes merely referred to as the DNAa of the present invention), theDNA encoding the protein b (hereinafter sometimes merely referred to asthe DNAb of the present invention), the antibody to the protein I,precursor protein I, partial peptide I, protein II, precursor proteinII, partial peptide II or signal peptide I, or salts thereof(hereinafter sometimes merely referred to as the antibody of the presentinvention) and the antisense DNA are explained with respect to theutilities. The protein a and protein b of the present invention aresometimes collectively referred to as the protein of the presentinvention, and the DNAa and DNAb of the present invention are sometimescollectively referred to the DNA of the present invention.

The protein a and protein b of the present invention can be utilized asdisease markers, since expression of these proteins increasestissue-specifically in the lung/bronchi in asthma model animals. Thatis, these proteins are useful as markers for early diagnosis inlung/chest diseases accompanied by inflammation of the lung/airways,judgment of severity in conditions, or predicted development ofdiseases.

(1) Therapeutic and Prophylactic Agent for the Diseases with which theProtein a of the Present Invention Is Associated

The protein a of the present invention is a member of the chitinasefamily. A chitinase is important for the biological protection mechanismagainst outward pathogens such as bacteria, virus, etc. Thus, theprotein a of the present invention or the DNAa of the present inventionmay be used as a therapeutic/prophylactic agent for various diseasesincluding immune diseases (e.g., autoimmune disease, immunodeficiency,allergic disease, etc.), infectious diseases (e.g., HIV (humanimmunodeficiency virus) infection, HBV (hepatitis B virus) infection,HCV (hepatitis C virus) infection, tuberculosis infection, opportunisticinfection, etc.), and the like.

When a patient has a reduced level of, or deficient in the protein a,etc. of the present invention in his or her body where the biologicalprotection mechanism is not exhibit sufficiently or normally, theprotein a of the present invention can provide its role sufficiently orproperly for the patient; (a) by administering the DNAa of the presentinvention to the patient to express the protein of the present inventionin the body, (b) by inserting the DNAa of the present invention into acell, expressing the protein of the present invention and thentransplanting the cell to the patient, (c) by administering the proteina of the present invention to the patient, or the like.

Where the DNAa of the present invention is used as thetherapeutic/prophylactic agents described above, the DNA alone isadministered directly to human or other warm-blooded animal;alternatively, the DNA is inserted into an appropriate vector such asretrovirus vector, adenovirus vector, adenovirus-associated virusvector, etc. and then administered to human or other warm-blooded animalin a conventional manner. The DNAa of the present invention may also beadministered as it is, or with adjuvants to assist its uptake by genegun or through a catheter such as a catheter with a hydrogel.

Where the protein a of the present invention is used as the aforesaidtherapeutic/prophylactic agents, it is preferred to use the same on apurified level of at least 90%, preferably at least 95%, more preferablyat least 98% and most preferably at least 99%.

The protein a of the present invention can be used orally, for example,in the form of tablets which maybe sugar coated if necessary, capsules,elixirs, microcapsules, etc., or parenterally in the form of injectablepreparations such as a sterile solution and a suspension in water orwith other pharmaceutically acceptable liquid. These preparations can beprepared by mixing the protein a of the present invention with aphysiologically acceptable known carrier, a flavoring agent, anexcipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc.in a unit dosage form required in a generally accepted manner that isapplied to making pharmaceutical preparations. The active ingredient inthe preparation is controlled in such a dose that an appropriate dose isobtained within the specified range given.

Additives miscible with tablets, capsules, etc. include a binder such asgelatin, corn starch, tragacanth and gum arabic, an excipient such ascrystalline cellulose, a swelling agent such as corn starch, gelatin andalginic acid, a lubricant such as magnesium stearate, a sweetening agentsuch as sucrose, lactose and saccharin, and a flavoring agent such aspeppermint, akamono oil or cherry. When the unit dosage is in the formof capsules, liquid carriers such as oils and fats may further be usedtogether with the additives described above. A sterile composition forinjection may be formulated according to a conventional manner used tomake a pharmaceutical, e.g., by dissolving or suspending the activeingredients in a vehicle such as water for injection with a naturallyoccurring vegetable oil such as sesame oil and coconut oil, etc. toprepare the pharmaceutical.

Examples of an aqueous medium for injection include physiological salineand an isotonic solution containing glucose and other auxiliary agents(e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used incombination with an appropriate dissolution aid such as an alcohol(e.g., ethanol or the like), a polyalcohol (e.g., propylene glycol andpolyethylene glycol), a nonionic surfactant (e.g., polysorbate 80™,HCO-50, etc.), and the like. Examples of the oily medium include sesameoil, soybean oil, etc., which may also be used in combination with adissolution aid such as benzyl benzoate, benzyl alcohol, etc. The agentmay further be formulated with a buffer (e.g., phosphate buffer, sodiumacetate buffer, etc.), a soothing agent (e.g., benzalkonium chloride,procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin,polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol,phenol, etc.), an antioxidant, etc. The thus-prepared liquid forinjection is normally filled in an appropriate ampoule.

The vector in which the DNAa of the present invention is inserted mayalso be prepared into pharmaceutical preparations in a manner similar tothe procedures above. Such preparations are generally used parenterally.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to human or otherwarm-blooded animal (e.g., rat, mouse, guinea pig, rabbit, chicken,sheep, swine, bovine, horse, cat, dog, monkey, chimpanzee, etc.).

The dose of the protein a of the present invention varies depending ontarget disease, subject to be administered, route for administration,etc.; for example, in oral administration for the treatment ofinfectious diseases, the dose is normally about 0.1 mg to about 100 mg,preferably about 1.0 to about 50 mg, and more preferably about 1.0 toabout 20 mg per day for adult (as 60 kg body weight). In parenteraladministration, the single dose varies depending on subject to beadministered, target disease, etc. but it is advantageous for thetreatment of infectious diseases to administer the protein intravenouslyat a daily dose of about 0.01 to about 30 mg, preferably about 0.1 toabout 20 mg, and more preferably about 0.1 to about 10 mg for adult (as60 kg body weight). For other animal species, the corresponding dose asconverted per 60 kg body weight can be administered.

(2) Screening of Drug Candidate Compounds for Disease

Because the protein a of the present invention belongs to the chitinasefamily, a compound or its salt capable of promoting the activities(e.g., a chitinase activity, etc.) of the protein a of the presentinvention can be used as medicaments for the treatment/prevention ofvarious diseases including immune diseases (e.g., autoimmune disease,immunodeficiency, allergic disease, etc.), infectious diseases (e.g.,HIV infection, HBV infection, HCV infection, tuberculosis infection,opportunistic infection, etc.), and the like.

On the other hand, the protein a of the present invention isincreasingly expressed prior to inflammation of the lung/bronchi, andcan thus be used as medicaments for the treatment/prevention oflung/chest diseases accompanied by inflammation of the lung/airways,including bronchial asthma, chronic obstructive pulmonary disease, etc.

Therefore, the protein a of the present invention is useful as a reagentfor screening the compound or its salts capable of promoting orinhibiting the activities of the protein a of the present invention.

That is, the present invention provides:

(1) a method for screening the compound or its salts capable ofpromoting the activities (e.g., a chitinase activity, etc.) of theprotein I, precursor protein I or partial peptide I, or its salts, ofthe present invention (hereinafter sometimes merely referred to as thepromoter), or the compound or its salts capable of inhibiting theactivities of the protein I, precursor protein I or partial peptide I,or its salts, of the present invention (hereinafter sometimes merelyreferred to as the inhibitor), which comprises using the protein I,precursor protein I or partial peptide I, or its salts, of the presentinvention. More specifically, the present invention provides, e.g.:

(2) a method for screening the promoter or the inhibitor, whichcomprises comparing (i) the case where a chitinase substrate is broughtinto contact with the protein I, precursor protein I or partial peptideI, or its salts, of the present invention and (ii) the case where achitinase substrate and a test compound are brought into contact withthe protein I, precursor protein I or partial peptide I, or its salts,of the present invention.

Specifically, in the screening method described above, the method ischaracterized by measuring, e.g., the chitinase activity of the proteina of the present invention in the cases (i) and (ii), and comparing thecases.

Examples of the substrate used are 4-methylumbelliferylβ-D-N,N′-diacetylchitobiose, 4-methylumbelliferylβ-D-N,N′,N″-triacetylchitobiose, p-nitrophenylβ-D-N,N′,N″-triacetylchitobiose, chitin azure, etc.

Examples of the test compound are a peptide, a protein, anon-peptidecompound, a synthetic compound, a fermentation product, a cell extract,a plant extract, an animal tissue extract and the like. These compoundsmay be novel compounds or publicly known compounds.

To perform the screening method described above, the protein a of thepresent invention is suspended in a buffer suitable for screening toprepare a specimen for the protein a of the present invention. Anybuffer having pH of approximately 4 to 10 (desirably a pH ofapproximately 6 to 8) such as a phosphate buffer, Tris-hydrochloridebuffer, etc. may be used, so long as it does not interfere the reactionbetween the protein a of the present invention and the substrate.

The chitinase activity of the protein a of the present invention can bedetermined by a publicly known method described in, e.g., J. Biol.Chem., 270, 2198 (1995), or its modification.

For example, when a test compound increases the chitinase activity in(ii) described above by at least about 20%, preferably at least 30%,more preferably at least about 50%, as compared to the case of (i)above, the test compound can be screened to be a compound capable ofpromoting the chitinase activity of the protein a of the presentinvention. On the other hand, a test compound can be screened to be acompound capable of inhibiting the chitinase activity of the protein aof the present invention, when the test compound inhibits the chitinaseactivity in (ii) described above by at least about 20%, preferably atleast 30%, more preferably at least about 50%, as compared to the caseof (i) above.

The kit for screening according to the present invention comprises theprotein I, precursor protein I or partial peptide I, or its salts, ofthe present invention.

The compounds or salts thereof obtained using the screening methods orscreening kits of the present invention are compounds screened: from thetest compounds described above, for example, peptides, proteins,non-peptide compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, animal tissue extracts, blood plasma, etc.,and are the compounds capable of promoting or inhibiting the activities(e.g., a chitinase activity, etc.) of the protein a of the presentinvention.

As salts of these compounds, there may be employed the same salts asthose of the protein I of the present invention described above.

The compounds capable of promoting the activities (e.g., a chitinaseactivity, etc.) of the protein a of the present invention can be used asmedicaments for the treatment/prevention of various diseases includingimmune diseases (e.g., autoimmune disease, immunodeficiency, allergicdisease, etc.), infectious diseases (e.g., HIV infection, HBV infection,HCV infection, tuberculosis infection, opportunistic infection, etc.),and the like.

On the other hand, the compounds capable of inhibiting the activities ofthe protein a of the present invention can be used as medicaments forthe treatment/prevention of lung/chest diseases accompanied byinflammation of the lung/airways, including bronchial asthma, chronicobstructive pulmonary disease, etc.

When the compounds obtained using the screening methods or screeningkits of the present invention are used as the therapeutic/prophylacticagents described above, they can be used in a conventional manner. Thecompounds maybe used, for example, in the form of tablets, capsules,elixirs, microcapsules, a sterile solution, a suspension, etc., as inthe pharmaceuticals containing the protein a of the present inventiondescribed above.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to human or otherwarm-blooded animal (e.g., mouse, rat, rabbit, sheep, swine, bovine,horse, chicken, cat, dog, monkey, chimpanzee, etc.).

The dose of the compound or salts thereof varies depending on itsaction, target disease, subject to be administered, route foradministration, etc.; when the compound capable of inhibiting theactivity of the protein a of the present invention is orallyadministered for the treatment of, e.g., bronchial asthma, the compoundis normally administered in a dose of about 0.1 to about 100 mg,preferably about 1.0 to about 50 mg, and more preferably about 1.0 toabout 20 mg per day for adult (as 60 kg body weight). In parenteraladministration, a single dose of the compound varies depending onsubject to be administered, target disease, etc. but it is advantageousfor the treatment of bronchial asthma to administer the compound capableof inhibiting the activity of the protein a of the present inventionintravenously in the form of injection in a daily dose of about 0.01 toabout 30 mg, preferably about 0.1 to about 20 mg, and more preferablyabout 0.1 to about 10 mg for adult (as 60 kg body weight). For otheranimal species, the corresponding dose as converted per 60 kg bodyweight can be administered.

On the other hand, when the compound capable of promoting the activityof the protein a of the present invention is orally administered for thetreatment of infectious diseases, the compound is normally administeredin a dose of about 0.1 to about 100 mg, preferably about 1.0 to about 50mg, and more preferably about 1.0 to about 20 mg per day for adult (as60 kg body weight). In parenteral administration, a single dose of thecompound varies depending on subject to be administered, target disease,etc. but it is advantageous for the treatment of infectious diseases toadminister the compound capable of promoting the activity of the proteina of the present invention intravenously in the form of injection in adaily dose of about 0.01 to about 30mg, preferably about 0.1 to about 20mg, and more preferably about 0.1 to about 10 mg for adult (as 60 kgbody weight) For other animal species, the corresponding dose asconverted per 60 kg body weight can be administered.

(3) Screening of Drug Candidate Compounds for the Diseases with whichthe Protein a or Protein b of the Present Invention Is Associated

The protein of the present invention is a secretory protein; forexample, the protein II is produced in the lung/airways of mouse asthmamodel prior to inflammation, and thus considered to be associated withinfiltration or activation of eoginophil, macrophage, etc. Therefore,the compound or its salt capable of inhibiting the activities of theprotein a or protein b of the present invention can be employed asmedicaments for the treatment/prevention of lung/chest diseasesaccompanied by inflammation of the lung/airways, including bronchialasthma, chronic obstructive pulmonary disease, etc. Thus, the protein ofthe present invention is useful as a reagent for screening the compoundor salts thereof capable of inhibiting the activities of the protein ofthe present invention.

That is, the present invention provides:

(1) a method for screening the compound capable of inhibiting theactivities (e.g., an eosinophil-mediated chemotactic activity, etc.) ofthe protein of the present invention (hereinafter sometimes merelyreferred to as the inhibitor, which comprises using the protein of thepresent invention. More specifically, the present invention provides,e.g.:

(2) a method for screening the inhibitor, which comprises comparing (i)the case where an eosinophil is brought into contact with the protein ofthe present invention and (ii) the case where an eosinophil and a testcompound are brought into contact with the protein of the presentinvention.

Specifically, in the screening method described above, the method ischaracterized by measuring, e.g., the eosinophil-mediated chemotacticactivity of the protein of the present invention in the cases (i) and(ii), and comparing them.

As the eosinophil, there is employed, e.g., mouse eosinophil, which canbe prepared by a publicly known method described in, e.g., J. LeukocyteBiol., 60, 573 (1996), or by a modification thereof.

Examples of the test compound are a peptide, a protein, a non-peptidecompound, a synthetic compound, a fermentation product, a cell extract,a plant extract, an animal tissue extract, and the like. These compoundsmay be novel compounds or publicly known compounds.

To perform the screening method described above, the protein of thepresent invention is suspended in a buffer suitable for screening toprepare a specimen for the protein of the present invention. Any bufferhaving pH of approximately 4 to 10 (desirably a pH of approximately 6 to8) such as a phosphate buffer, Tris-hydrochloride buffer, etc. may beused, so long as it does not interfere the chemotactic reaction ofeosinophils.

The eosinophil-mediated chemotactic activity of the protein of thepresent invention can be determined by a publicly known method describedin, e.g., Immunity, 4, 1 (1996), or its modification.

For example, when a test compound increases the eosinophil-mediatedchemotactic activity in the case (ii) described above by at least about20%, preferably at least 30%, more preferably at least about 50%, ascompared to the case of (i) above, the test compound can be screened tobe a compound capable of inhibiting the eosinophil-mediated chemotacticactivity of the protein of the present invention.

The compounds or salts thereof obtained using the screening methods orscreening kits of the present invention are compounds screened from thetest compounds described above, for example, peptides, proteins,non-peptide compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, animal tissue extracts, blood plasma, etc.,and are the compounds capable of inhibiting the activities (e.g., aneosinophil-mediated chemotactic activity, etc.) of the protein of thepresent invention.

As salts of these compounds, there may be employed the same salts asthose of the protein I of the present invention described above.

The compound capable of inhibiting the activities of the protein of thepresent invention is useful as medicaments for the treatment/preventionof lung/chest diseases accompanied by inflammation of the lung/airways,including bronchial asthma, chronic obstructive pulmonary disease, etc.

When the compounds obtained using the screening methods or screeningkits of the present invention are used as the therapeutic/prophylacticagents described above, they can be used in a conventional manner. Thecompounds may be used, for example, in the form of tablets, capsules,elixirs, microcapsules, a sterile solution, a suspension, etc., as inthe pharmaceuticals containing the protein a of the present inventiondescribed above.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to human or otherwarm-blooded animal (e.g., mouse, rat, rabbit, sheep, swine, bovine,horse, chicken, cat, dog, monkey, chimpanzee, etc.).

The dose of the compound or salts thereof varies depending on itsaction, target disease, subject to be administered, route foradministration, etc.; when the compound capable of inhibiting theactivity of the protein of the present invention is orally administeredfor the treatment of, e.g., bronchial asthma, the compound is normallyadministered in a dose of about 0.1 to about 100 mg, preferably about1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per dayfor adult (as 60 kg body weight). In parenteral administration, a singledose of the compound varies depending on subject to be administered,target disease, etc. but it is advantageous for the treatment ofbronchial asthma to administer the compound capable of inhibiting theactivity of the protein of the present invention intravenously in theform of injection in a daily dose of about 0.01 to about 30 mg,preferably about 0.1 to about 20 mg, and more preferably about 0.1 toabout 10 mg for adult (as 60 kg body weight). For other animal species,the corresponding dose as converted per 60 kg body weight can beadministered.

(4) Quantification for the Protein a or Protein b of the Invention

The antibody to the protein of the present invention (hereinaftersometimes merely referred to as the antibody of the present invention)is capable of specifically recognizing the protein of the presentinvention, and can thus be used for a quantification of the protein ofthe present invention in a test sample fluid, in particular, for aquantification by sandwich immunoassay.

That is, the present invention provides:

(i) a method for quantification of the protein of the present inventionin a test sample fluid, which comprises competitively reacting theantibody of the present invention, a test sample fluid and the labeledprotein of the present invention, and measuring the ratio of the labeledprotein of the present invention bound to said antibody; and,

(ii) a method for quantification of the protein of the present inventionin a test sample fluid, which comprises reacting the test sample fluidsimultaneously or continuously with the antibody of the presentinvention immobilized on a carrier and another labeled antibody of thepresent invention, and then measuring the activity of the labeling agenton the insoluble carrier.

In the method (ii) for quantification described above, it is preferredthat one antibody is capable of recognizing the N-terminal region of theprotein of the present invention (preferably the protein I or protein IIof the present invention), while another antibody is capable ofrecognizing the C-terminal region of the protein of the presentinvention (preferably the protein I or protein II of the presentinvention).

The monoclonal antibody to the protein of the present invention(hereinafter sometimes referred to as the monoclonal antibody of theinvention) may be used to assay the protein of the present invention.Moreover, the protein can be detected by means of a tissue staining aswell. For these purposes, the antibody molecule per se may be used orF(ab′)₂, Fab′ or Fab fractions of the antibody molecule may also beused.

There is no particular limitation to the method for quantification ofthe protein of the present invention using the antibody of the presentinvention; any method may be used, so far as it relates to a method, inwhich the amount of antibody, antigen or antibody-antigen complex can bedetected by a chemical or physical means, depending on or correspondingto the amount of antigen (e.g., the amount of a protein) in a testsample fluid to be assayed, and then calculated using a standard curveprepared by a standard solution containing the known amount of antigen.Advantageously used are, for example, nephrometry, competitive method,immunometric method and sandwich method; in terms of sensitivity andspecificity, the sandwich method, which will be described later, isparticularly preferred.

Examples of the labeling agent used in the assay method using thelabeling substance are radioisotopes, enzymes, fluorescent substances,luminescent substances, and the like. Examples of the radioisotope are[¹²⁵¹I], [¹³¹I], [³H], [¹⁴C], etc. Preferred examples of the enzyme arethose that are stable and have a high specific activity, which includeβ-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, etc. Examples of the fluorescent substance arefluorescamine, fluorescein isothiocyanate, etc. Examples of theluminescent substance are luminol, a luminol derivative, luciferin,lucigenin, etc. Furthermore, the biotin-avidin system may also be usedfor binding of an antibody or antigen to a labeling agent.

In the immobilization of antigens or antibodies, physical adsorption maybe used. Alternatively, chemical binding that is conventionally used forimmobilization of proteins or enzymes may be used as well. Examples ofthe carrier include insoluble polysaccharides such as agarose, dextranand cellulose; synthetic resins such as polystyrene, polyacrylamide andsilicone; glass; etc.

In the sandwich method, a test sample fluid is reacted with animmobilized monoclonal antibody of the present invention (firstreaction), then reacted with another labeled monoclonal antibody of thepresent invention (second reaction) and the activity of the labelingagent on the insoluble carrier is assayed, whereby the amount of theprotein of the present invention in the test sample fluid can bequantified. The first and second reactions may be carried out in areversed order, simultaneously or sequentially with an interval. Thetype of the labeling agent and the method for immobilization may be thesame as those described hereinabove. In the immunoassay by the sandwichmethod, it is not always necessary that the antibody used for thelabeled antibody and for the solid phase should be one type or onespecies but a mixture of two or more antibodies may also be used for thepurpose of improving the measurement sensitivity, etc.

In the method according to the present invention for assaying theprotein of the present invention by the sandwich method, preferredmonoclonal antibodies of the present invention used for the first andthe second reactions are antibodies, which binding sites to the proteinof the present invention are different from one another. That is, theantibodies used in the first and the second reactions are those wherein,when the antibody used in the second reaction recognizes the C-terminalregion of the protein of the present invention, the antibody recognizingthe site other than the C-terminal regions, e.g., recognizing theN-terminal region, is preferably used in the first reaction.

The monoclonal antibody of the present invention may be used in an assaysystem other than the sandwich method, such as a competitive method, animmunometric method, a nephrometry, etc.

In the competitive method, an antigen in a test sample fluid and alabeled antigen are competitively reacted with an antibody, then theunreacted labeled antigen (F) and the labeled antigen bound to theantibody (B) are separated (i.e., B/F separation) and the labeled amountof either B or F is measured to determine the amount of the antigen inthe test sample fluid. In the reactions for such a method, there are aliquid phase method in which a soluble antibody is used as the antibodyand the B/F separation is effected by polyethylene glycol while a secondantibody to the antibody is used, and a solid phase method in which animmobilized antibody is used as the first antibody or a soluble antibodyis used as the first antibody while an immobilized antibody. is used asthe second antibody.

In the immunometric method, an antigen in a test sample fluid and animmobilized antigen are competitively reacted with a given amount of alabeled antibody followed by separating the solid phase from the liquidphase; or an antigen in a test sample fluid and an excess amount oflabeled antibody are reacted, then an immobilized antigen is added tobind an unreacted labeled antibody to the solid phase and the solidphase is separated from the liquid phase. Thereafter, the labeled amountof any of the phases is measured to determine the antigen amount in thetest sample fluid.

In the nephrometry, the amount of insoluble sediment, which is producedas a result of the antigen-antibody reaction in a gel or in a solution,is measured. Even when the amount of an antigen in a test sample fluidis small and only a small amount of the sediment is obtained, a lasernephrometry utilizing laser scattering can be suitably used.

In applying each of those immunoassays to the assay method for thepresent invention, any special conditions or operations are not requiredto set forth. The assay system for the protein of the present inventionmay be constructed in addition to conditions or operationsconventionally used for each of the methods, taking the technicalconsideration of one skilled in the art into account consideration. Forthe details of such conventional technical means, a variety of reviews,reference books, etc. may be referred to.

For example, there are Hiroshi Irie (ed.): “Radioimmunoassay” (publishedby Kodansha, 1974); Hiroshi Irie (ed.): “Radioimmunoassay; SecondSeries” (published by Kodansha, 1979); Eiji Ishikawa, et al. (ed.):“Enzyme Immunoassay” (published by Igaku Shoin, 1978); Eiji Ishikawa, etal. (ed.): “Enzyme Immunoassay” (Second Edition) (published by IgakuShoin, 1982); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (ThirdEdition) (published by Igaku Shoin, 1987); “Methods in Enzymology” Vol.70 (Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (published by Academic Press); etc.)

As described above, the protein of the present invention can bequantified with high sensitivity, using the antibody of the presentinvention.

Furthermore, (1) when an increased level of the protein of the presentinvention is detected by quantifying the level of the protein of thepresent invention using the antibody of the present invention, it can bediagnosed that one suffers from diseases such as lung/chest diseasesaccompanied by inflammation of the lung/airways, including bronchialasthma, chronic obstructive pulmonary disease, etc., or it is highlylikely to suffer from these disease in the future.

The antibody of the present invention can be employed for detecting theprotein of the present invention, which is present in a test samplefluid such as a body fluid, a tissue, etc. The antibody can also be usedto prepare an antibody column for purification of the protein of thepresent invention, detect the protein of the present invention in eachfraction upon purification, and analyze the behavior of the protein ofthe present invention in the cells under investigation.

(5) Gene Diagnostic Agent

By using the DNA of the present invention, e.g., as a probe, anabnormality (gene abnormality) of the DNA or mRNA encoding the proteinof the present invention in human or warm-blooded animal (e.g., rat,mouse, guinea pig, rabbit, chicken, sheep, swine, bovine, horse, cat,dog, monkey, chimpanzee, etc.) can be detected. Therefore, the DNA ofthe present invention is useful as a gene diagnostic agent for detectingdamages to the DNA or mRNA, its mutation, or decreased expression,increased expression, over expression, etc. of the DNA or mRNA.

The gene diagnosis described above using the DNA of the presentinvention can be performed by, for example, the publicly known Northernhybridization assay or the PCR-SSCP assay (Genomics, 5, 874-879 (1989);Proceedings of the National Academy of Sciences of the United States ofAmerica, 86, 2766-2770 (1989)), etc.

In case that overexpression is detected by, e.g., the Northernhybridization or DNA mutation is detected by the PCR-SSCP assay, it canbe diagnosed that it is highly likely to suffer from diseases such aslung/chest diseases accompanied by inflammation of the lung/airwaysincluding bronchial asthma, chronic obstructive pulmonary disease, etc.

(6) Pharmaceutical Comprising an Antisense DNA

An antisense DNA that binds to the DNA of the present inventioncomplementarily to suppress expression of the DNA can be used as theagent for the treatment/prevention of diseases such as lung/chestdiseases accompanied by inflammation of the lung/airways includingbronchial asthma, chronic obstructive pulmonary disease, etc., since theantisense DNA can suppress production of the protein of the presentinvention in vivo.

In the case that the antisense DNA described above is used as thetherapeutic/prophylactic agent, the therapeutic/prophylactic agents forvarious diseases described above comprising the DNA of the presentinvention apply similarly to the antisense DNA.

For example, when the antisense DNA is used, the antisense DNA isadministered directly, or the antisense DNA is inserted into anappropriate vector such as retrovirus vector, adenovirus vector,adenovirus-associated virus vector, etc., followed by treating in aconventional manner. The antisense DNA may be administered as it stands,or with a physiologically acceptable carrier to assist its uptake bygene gun or through a catheter such as a catheter with a hydrogel.Alternatively, the antisense DNA may be prepared into an aerosol, whichis locally administered into the trachea as an inhalant.

In addition, the antisense DNA may also be employed as anoligonucleotide probe for diagnosis to examine the presence of the DNAof the present invention in tissues or cells and states of itsexpression.

(7) Pharmaceutical Composition Comprising the Antibody of the PresentInvention

The DNA of the present invention having an activity of neutralizing theprotein of the present invention can be used as a medicament fordiseases such as lung/chest diseases accompanied by inflammation of thelung/airways including bronchial asthma, chronic obstructive pulmonarydisease, etc.

The aforesaid therapeutic/prophylactic agent containing the antibody ofthe invention for the diseases described above can be administeredorally or parenterally to human or other warm-blooded animal (e.g., rat,rabbit, sheep, swine, bovine, cat, dog, monkey, etc.), in its liquidform as it stands, or as a pharmaceutical composition in a suitablepreparation form. The dose varies depending on subject to beadministered, target disease, condition, route for administration, etc.;when the agent is administered to adult for the treatment/prevention of,e.g., bronchial asthma, the antibody of the present invention isnormally advantageously administered intravenously, about 1to about 1times a day, preferably about 1 to 3 times a day, in a single dose ofabout 0.01 to about 20 mg/kg body weight, preferably about 0.1 to about10 mg/kg body weight, and more preferably about 0.1 to about 5 mg/kgbody weight for adult. For other parenteral administration and oraladministration, the dose corresponding to the dose above can beadministered; when the condition is especially severe, the dose may beincreased accordingly to the condition.

The antibody of the present invention may be administered in itself oras an appropriate pharmaceutical composition. The pharmaceuticalcomposition used for the administration described above contains apharmacologically acceptable carrier with the aforesaid compounds orsalts thereof, a diluent or excipient. Such a composition is provided inthe preparation suitable for oral or parenteral administration.

That is, examples of the composition for oral administration includesolid or liquid preparations, specifically, tablets (including drageesand film-coated tablets), pills, granules, powdery preparations,capsules (including soft capsules), syrup, emulsions, suspensions, etc.Such a composition is manufactured by publicly known methods andcontains a vehicle, a diluent or an excipient conventionally used in thefield of pharmaceutical preparations. Examples of the vehicle orexcipient for tablets are lactose, starch, sucrose, magnesium stearate,etc.

Examples of the composition for parenteral administration that can beused are injections, suppositories, etc. and the injections include theform of intravenous, subcutaneous, transcutaneous, intramuscular anddrip injections, etc. Such injections are prepared by publicly knownmethods, e.g., by dissolving, suspending or emulsifying the aforesaidantibody or its salts in a sterile aqueous or oily liquid medium. Forthe aqueous medium for injection, for example, physiological saline andisotonic solutions containing glucose and other adjuvant, etc. are used.Appropriate dissolution aids, for example, alcohol (e.g., ethanol),polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionicsurfactant (e.g., polysorbate 80™, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)) may be used in combination. For theoily solution, for example, sesame oil, soybean oil and the like areused, and dissolution aids such as benzyl benzoate and benzyl alcoholmay be used in combination. The thus-prepared liquid for injection isnormally filled in an appropriate ampoule. The suppository used forrectal administration is prepared by mixing the aforesaid antibody orits salts with conventional suppository base.

The oral or parenteral pharmaceutical composition described above isadvantageously prepared in a unit dosage form suitable for the dose ofthe active ingredient. Examples of such unit dosage form includetablets, pills, capsules, injections (ampoules), suppositories, etc. Itis preferred that the antibody described above is contained generally ina dose of 5 to 500 mg per unit dosage form, 5 to 100 mg especially forinjections and 10 to 250 mg for other preparations.

Each composition described above may further contain other activecomponents unless formulation with the antibody causes any adverseinteraction.

(8) DNA Transgenic Animal

The present invention provides a non-human mammal bearing DNA encodingthe protein of the present invention, which is exogenous (hereinafterabbreviated as the exogenous DNA of the present invention) or itsvariant DNA (sometimes simply referred to as the exogenous variant DNAof the present invention).

Thus, the present invention provides:

(1) a non-human mammal bearing the exogenous DNA of the presentinvention or its variant DNA;

(2) the mammal according to (1), wherein the non-human mammal is arodent;

(3) the mammal according to (2), wherein the rodent is mouse or rat;and,

(4) a recombinant vector bearing the exogenous DNA of the presentinvention or its variant DNA and capable of expressing in a mammal.

The non-human mammal bearing the exogenous DNA of the present inventionor its variant DNA (hereinafter simply referred to as the DNA transgenicanimal of the present invention) can be created by transfecting adesired DNA into an unfertilized egg, a fertilized egg, a spermatozoon,a germinal cell containing a primordial germinal cell thereof, or thelike, preferably in the embryogenic stage in the development of anon-human mammal (more preferably in the single cell or fertilized cellstage and generally before the 8-cell phase), by standard means, such asthe calcium phosphate method, the electric pulse method, the lipofectionmethod, the agglutination method, the microinjection method, theparticle gun method, the DEAE-dextran method, etc. Also, it is possibleto transfect the exogenous DNA of the present invention into a somaticcell, a living organ, a tissue cell, or the like by the DNA transfectionmethods, and utilize the transformant for cell culture, tissue culture,etc. In addition, these cells maybe fused with the above-describedgerminal cell by a publicly known cell fusion method to create the DNAtransgenic animal of the present invention.

Examples of the non-human mammal that can be used include bovine, swine,sheep, goat, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, andthe like. Above all, preferred are rodents, especially mice (e.g.,C57Bl/6 strain, DBA2 strain, etc. for the pure line and for the crossline, B6C3F₁ strain, BDF₁ strain B6D2F₁ strain, BALB/c strain, ICRstrain, etc.) or rats (e.g., Wistar, SD, etc.), since they arerelatively short in ontogeny and life cycle from a standpoint ofcreating model animals for human disease.

“Mammals” in a recombinant vector that can be expressed in the mammalsinclude the aforesaid non-human mammals and human.

The exogenous DNA of the present invention refers to the DNA of thepresent invention that is once isolated and extracted from mammals, notthe DNA of the present invention inherently possessed by the non-humanmammals.

The variant DNA of the present invention includes mutants resulting fromvariation (e.g., mutation, etc.) in the base sequence of the originalDNA of the present invention, specifically DNAs resulting from baseaddition, deletion, substitution with other bases, etc. and furtherincluding abnormal DNA.

The abnormal DNA is intended to mean a DNA that expresses the abnormalprotein of the present invention and exemplified by a DNA that expressesa protein to suppress the functions of the normal protein of the presentinvention.

The exogenous DNA of the present invention may be any one of thosederived from a mammal of the same species as, or a different speciesfrom, the mammal as the target animal. In transfecting the DNA of thepresent invention, it is generally advantageous to use the DNA as a DNAconstruct in which the DNA is ligated downstream a promoter capable ofexpressing the DNA in the target animal. For example, in the case oftransfecting the human DNA of the present invention, a DNA transgenicmammal that expresses the DNA of the present invention to a high level,can be prepared by microinjecting a DNA construct (e.g., vector, etc.)ligated with the human DNA of the present invention into a fertilizedegg of the target non-human mammal downstream various promoters whichare capable of expressing the DNA derived from various mammals (e.g.,rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) bearingthe DNA of the present invention highly homologous to the human DNA.

As expression vectors for the protein of the present invention, thereare Escherichia coli-derived plasmids, Bacillus subtilis-derivedplasmids, yeast-derived plasmids, bacteriophages such as λ phage,retroviruses such as Moloney leukemia virus, etc., and animal virusessuch as vaccinia virus, baculovirus, etc. Of these vectors, Escherichiacoli-derived plasmids, Bacillus subtilis-derived plasmids, oryeast-derived plasmids, etc. are preferably used.

Examples of these promoters for regulating the DNA expression describedabove include 1) promoters for DNA derived from viruses (e.g., simianvirus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancervirus, poliovirus, etc.), and 2) promoters derived from various mammals(human, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.),for example, promoters of albumin, insulin II, uroplakin II, elastase,erythropoietin, endothelin, muscular creatine kinase, glial fibrillaryacidic protein, glutathione S-transferase, platelet-derived growthfactor β, keratins K1, K10 and K14, collagen types I and II, cyclicAMP-dependent protein kinase βI subunit, dystrophin, tartarate-resistantalkaline phosphatase, atrial natriuretic factor, endothelial receptortyrosine kinase (generally abbreviated as Tie2), sodium-potassiumadenosine triphosphorylase (Na, K-ATPase), neurofilament light chain,metallothioneins I and IIA, metalloproteinase I tissue inhibitor, MHCclass I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroidperoxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), βactin, α and β myosin heavy chains, myosin light chains 1 and 2, myelinbase protein, thyroglobulins, Thy-1, immunoglobulins, H-chain variableregion (VNP), serum amyloid component P, myoglobin, troponin C, smoothmuscle α actin, preproencephalin A, vasopressin, etc. Among them,cytomegalovirus promoters, human polypeptide elongation factor 1α(EF-1α) promoters, human and chicken β actin promoters, etc., whichprotein can highly express in the whole body are preferred.

It is preferred that the vectors described above have a sequence forterminating the transcription of the desired messenger RNA in the DNAtransgenic animal (generally termed terminator); for example, a sequenceof each DNA derived from viruses and various mammals. SV40 terminator ofthe simian virus, etc. are preferably used.

In addition, for the purpose of increasing the expression of the desiredexogenous DNA to a higher level, the splicing signal and enhancer regionof each DNA, a portion of the intron of an eukaryotic DNA may also beligated at the 5′ upstream of the promoter region, or between thepromoter region and the translational region, or at the 3′ downstream ofthe translational region, depending upon purposes.

The translational region for the normal protein of the present inventioncan be obtained using as a starting material the entire genomic DNA orits portion of liver, kidney, thyroid cell or fibroblast origin fromhuman or various mammals (e.g., rabbits, dogs, cats, guinea pigs,hamsters, rats, mice, etc.) or of various commercially available genomicDNA libraries, or using complementary DNA prepared by a publicly knownmethod from RNA of liver, kidney, thyroid cell or fibroblast origin as astarting material. Also, an exogenous abnormal DNA can be obtained usingcomplementary DNA prepared by a publicly known method from RNA of humanfibroblast origin as a starting material. Alternatively, thetranslational region for a normal protein translational region obtainedby the cell or tissue described above can be made variant by pointmutagenesis.

The translational region can be prepared by a conventional geneticengineering technique, in which the DNA is ligated downstream theaforesaid promoter and if desired, upstream the translation terminationsite, as a DNA construct capable of being expressed in the transgenicanimal.

The exogenous DNA of the present invention is transfected at thefertilized egg cell stage in a manner such that the DNA is certainlypresent in all the germinal cells and somatic cells of the targetmammal. The fact that the exogenous DNA of the present invention ispresent in the germinal cells of the animal prepared by DNA transfectionmeans that all offspring of the prepared animal will maintain theexogenous DNA of the present invention in all of the germinal cells andsomatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention also have the exogenous DNA ofthe present invention in all of the germinal cells and somatic cellsthereof.

The non-human mammal in which the normal exogenous DNA of the presentinvention has been transfected can be passaged as the DNA-bearing animalunder ordinary rearing environment, by confirming that the exogenous DNAis stably retained by mating.

By the transfection of the exogenous DNA of the present invention at thefertilized egg cell stage, the DNA is retained to be excess in all ofthe germinal and somatic cells. The fact that the exogenous DNA of thepresent invention is excessively present in the germinal cells of theprepared animal after transfection means that the exogenous DNA of thepresent invention is excessively present in all of the germinal cellsand somatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention have excessively the DNA of thepresent invention in all of the germinal cells and somatic cellsthereof.

By obtaining a homozygotic animal having the transfected DNA in both ofhomologous chromosomes and mating a male and female of the animal, alloffspring can be passaged to retain the DNA.

In a non-human mammal bearing the normal DNA of the present invention,the normal DNA of the present invention has expressed to a high level,and may eventually develop the accentuated function of the protein ofthe present invention by promoting the function of endogenous normalDNA. Therefore, the animal can be utilized as a pathologic model animalfor such a disease. For example, using the normal DNA transgenic animalof the present invention, it is possible to elucidate the mechanism ofthe accentuated function of the protein of the present invention and thepathological mechanism of the disease associated with the protein of thepresent invention and to determine how to treat the disease.

Furthermore, since a mammal transfected the exogenous normal DNA of thepresent invention exhibits a symptom of increasing the protein of thepresent invention liberated, the animal is usable for screening thetherapeutic agent for the disease associated with the protein of thepresent invention.

On the other hand, non-human mammal having the exogenous abnormal DNA ofthe present invention can be passaged under normal breeding conditionsas the DNA-bearing animal by confirming the stable retaining of theexogenous DNA via crossing. Furthermore, the desired exogenous DNA canbe utilized as a starting material by inserting the DNA into the plasmiddescribed above. The DNA construct with a promoter can be prepared byconventional genetic engineering techniques. The transfection of theabnormal DNA of the present invention at the fertilized egg cell stageis preserved to be present in all of the germinal and somatic cells ofthe mammals to be subjected. The fact that the abnormal DNA of thepresent invention is present in the germinal cells of the animal afterDNA transfection means that all of the offspring of the prepared animalhave the abnormal DNA of the present invention in all of the germinaland somatic cells. Such an offspring passaged the exogenous DNA of thepresent invention contains the abnormal DNA of the present invention inall of the germinal and somatic cells. A homozygous animal having theintroduced DNA on both of homologous chromosomes can be acquired andthen by mating these male and female animals, all the offspring can bebled to have the DNA.

Since non-human mammal having the abnormal DNA of the present inventionmay express the abnormal DNA of the present invention at a high level,the animal may be the function inactivation type in adaptability of theprotein of the present invention by inhibiting the function of theendogenous normal DNA and can be utilized as its disease model animal.For example, using the abnormal DNA-transgenic animal of the presentinvention, it is possible to elucidate the mechanism of in adaptabilityof the protein of the present invention and to perform to study a methodfor treatment of this disease.

More specifically, the transgenic animal expressing the abnormal DNA ofthe present invention to a high level is also expected to serve as anexperimental model for the elucidation of the mechanism of thefunctional inhibition (dominant negative effect) of normal protein bythe abnormal protein of the present invention in the function inactivetype in adaptability of the protein of the present invention.

A mammal bearing the abnormal exogenous DNA of the present invention isalso expected to serve for screening a candidate drug for the treatmentof the function inactive type in adaptability of the protein of thepresent invention, since the protein of the present invention isincreased in such an animal in its free form.

Other potential applications of two kinds of the transgenic animalsdescribed above include:

1) use as a cell source for tissue culture;

2) elucidation of the relation to a protein that is specificallyexpressed or activated by the protein of the present invention, bydirect analysis of DNA or RNA in tissue of the DNA transgenic animal ofthe present invention or by analysis of the protein tissue expressed bythe DNA;

3) research in the function of cells derived from tissues that arecultured usually only with difficulty, using cells of tissue bearing theDNA cultured by a standard tissue culture technique;

4) screening of a drug that enhances the functions of cells using thecells described in 3) above; and,

5) isolation and purification of the variant protein of the presentinvention and preparation of an antibody thereto.

Furthermore, clinical conditions of a disease associated wit the proteinof the present invention, including the function inactive type inadaptability of the protein of the present invention can be determinedusing the DNA transgenic animal of the present invention. Also,pathological findings on each organ in a disease model associated withthe protein of the present invention can be obtained in more detail,leading to the development of a new method for treatment as well as theresearch and therapy of any secondary diseases associated with thedisease.

It is also possible to obtain a free DNA-transfected cell by withdrawingeach organ from the DNA transgenic animal of the present invention,mincing the organ and degrading with a proteinase such as trypsin, etc.,followed by establishing the line of culturing or cultured cells.Furthermore, the DNA transgenic animal of the present invention canserve as identification of cells capable of producing the protein of thepresent invention, and as studies on association with apoptosis,differentiation or propagation or on the mechanism of signaltransduction in these properties to inspect any abnormality therein.Thus the DNA transgenic animal of the present invention can provide aneffective research material for the protein of the present invention andfor elucidating the function and effect thereof.

To develop a drug for the treatment of diseases associated with theprotein of the present invention, including the function inactive typein adaptability of the protein of the present invention, using the DNAtransgenic animal of the present invention, an effective and rapidmethod for screening can be provided by using the method for inspectionand the method for quantification, etc. described above. It is alsopossible to investigate and develop a method for DNA therapy for thetreatment of diseases associated with the protein of the presentinvention, using the DNA transgenic animal of the present invention or avector capable of expressing the exogenous DNA of the present invention.

(9) Knockout Animal

The present invention provides a non-human mammal embryonic stem cellbearing the DNA of the present invention inactivated and a non-humanmammal deficient in expressing the DNA of the present invention.

Thus, the present invention provides:

(1) a non-human embryonic stem cell, in which the DNA of the presentinvention is inactivated;

(2) an embryonic stem cell according to (1), wherein the DNA isinactivated by introducing a reporter gene (e.g., β-galactosidase genederived from Escherichia coli);

(3) an embryonic stem cell according to (1), which is resistant toneomycin;

(4) an embryonic stem cell according to (1), wherein the non-humanmammal is a rodent;

(5) an embryonic stem cell according to (4), wherein the rodent ismouse;

(6) a non-human mammal deficient in expressing the DNA of the presentinvention, wherein the DNA is inactivated;

(7) a non-human mammal according to (6), wherein the DNA is inactivatedby inserting a reporter gene (e.g., β-galactosidase derived fromEscherichia coli) therein and the reporter gene is capable of beingexpressed under control of a promoter for the DNA of the presentinvention;

(8) a non-human mammal according to (6), wherein the non-human mammal isa rodent;

(9) a non-human mammal according to (8), wherein the rodent is mouse;and,

(10) a method for screening a compound capable of promoting orinhibiting the promoter activity for the DNA of the present invention,which comprises administering a test compound to the mammal of (7) anddetecting expression of the reporter gene.

The non-human mammal embryonic stem cell, in which the DNA of thepresent invention is inactivated, refers to a non-human mammal embryonicstem cell that suppresses the ability of the non-human mammal to expressthe DNA by artificially mutating the DNA of the present invention, orthe DNA has no substantial ability to express the protein of the presentinvention (hereinafter sometimes referred to as the knockout DNA of thepresent invention) by substantially inactivating the activities of theprotein of the present invention encoded by the DNA (hereinafter merelyreferred to as ES cell).

As the non-human mammal, the same examples as described above apply.

Techniques for artificially mutating the DNA of the present inventioninclude deletion of a part or all of the DNA sequence and insertion ofor substitution with other DNA, by genetic engineering. By thesevariations, the knockout DNA of the present invention may be prepared,for example, by shifting the reading frame of a codon or by disruptingthe function of a promoter or exon.

Specifically, the non-human mammal embryonic stem cell in which the DNAof the present invention is inactivated (hereinafter merely referred toas the ES cell with the DNA of the present invention inactivated or theknockout ES cell of the present invention) can be obtained by, forexample, isolating the DNA of the present invention that the desirednon-human mammal possesses, inserting a DNA fragment having a DNAsequence constructed by inserting a drug resistant gene such as aneomycin resistant gene or a hygromycin resistant gene, or a reportergene such as lacZ (β-galactosidase gene) or cat (chloramphenicolacetyltransferase gene), etc. into its exon site thereby to disable thefunctions of exon, or integrating to a chromosome of the subject animalby, e.g., homologous recombination, a DNA sequence which terminates genetranscription (e.g., poly A additional signal, etc.) in the intronbetween exons thus to inhibit the synthesis of complete messenger RNAand eventually destroy the gene (hereinafter simply referred to astargeting vector). The thus obtained ES cells is subjected to Southernhybridization analysis with a DNA sequence on or near the DNA of thepresent invention as a probe, or to PCR analysis with a DNA sequence onthe targeting vector and another DNA sequence near the DNA of thepresent invention, which is not included in the targeting vector asprimers, to select the knockout ES cell of the present invention.

The parent ES cells to inactivate the DNA of the present invention byhomologous recombination, etc. may be of a strain already established asdescribed above, or may be originally established in accordance with amodification of the known method by Evans and Kaufman supra. Forexample, in the case of mouse ES cells, currently it is common practiceto use ES cells of the 129 strain. However, since their immunologicalbackground is obscure, the C57BL/6 mouse or the BDF₁ mouse (F1 hybridbetween C57BL/6 and DBA/2), wherein the low ovum availability perC57BL/6 in the C57BL/6 mouse has been improved by crossing with DBA/2,may be preferably used, instead of obtaining a pure line of ES cellswith the clear immunological genetic background and for other purposes.The BDF₁ mouse is advantageous in that, when a pathologic model mouse isgenerated using ES cells obtained therefrom, the genetic background canbe changed to that of the C57BL/6 mouse by back-crossing with theC57BL/6 mouse, since its background is of the C57BL/6 mouse, as well asbeing advantageous in that ovum availability per animal is high and ovaare robust.

In establishing ES cells, blastocytes at 3.5 days after fertilizationare commonly used. In the present invention, embryos are preferablycollected at the 8-cell stage, after culturing until the blastocytestage, the embryos are used to efficiently obtain a large number ofearly stage embryos.

Although the ES cells used may be of either sex, male ES cells aregenerally more convenient for generation of a germ cell line chimera andare therefore preferred. It is also desirable that sexes be identifiedas soon as possible to save painstaking culture time.

Methods for sex identification of the ES cell include the method inwhich a gene in the sex-determining region on the Y-chromosome isamplified by the PCR process and detected. When this method is used, onecolony of ES cells (about 50 cells) is sufficient for sex-determinationanalysis, which karyotype analysis, for example G-banding method,requires about 10⁶ cells; therefore, the first selection of ES cells atthe early stage of culture can be based on sex identification, and malecells can be selected early, which saves a significant amount of time atthe early stage of culture.

Second selection can be achieved by, for example, number of chromosomeconfirmation by the G-banding method. It is usually desirable that thechromosome number of the obtained ES cells be 100% of the normal number.However, when it is difficult to obtain the cells having the normalnumber of chromosomes due to physical operation etc. in cellestablishment, it is desirable that the ES cell be again cloned to anormal cell (e.g., in mouse cells having the number of chromosomes being2n=40) after the gene of the ES cells is rendered knockout.

Although the embryonic stem cell line thus obtained shows a very highgrowth potential, it must be subcultured with great care, since it tendsto lose its ontogenic capability. For example, the embryonic stem cellline is cultured at about 37° C. in a carbon dioxide incubator(preferably about 5% carbon dioxide and about 95% air, or about 5%oxygen, about 5% carbon dioxide and 90% air) in the presence of LIF (1to 10000 U/ml) on appropriate feeder cells such as STO fibroblasts,treated with a trypsin/EDTA solution (normally about 0.001 to about 0.5%trypsin/about 0.1 to about 5 mM EDTA, preferably about 0.1% trypsin/1 mMEDTA) at the time of passage to obtain separate single cells, which arethen seeded on freshly prepared feeder cells. This passage is normallyconducted every 1 to 3 days; it is desirable that cells be observed atpassage and cells found to be morphologically abnormal in culture, ifany, be abandoned.

Where ES cells are allowed to reach a high density in mono-layers or toform cell aggregates in suspension under appropriate conditions, theywill spontaneously differentiate to various cell types, for example,pariental and visceral muscles, cardiac muscle or the like [M. J. Evansand M. H. Kaufman, Nature, 292, 154, 1981; G. R. Martin, Proc. Natl.Acad. Sci. U.S.A., 78, 7634, 1981; T. C. Doetschman et al., Journal ofEmbryology Experimental Morphology, 87, 27, 1985]. The cells deficientin expression of the DNA of the present invention, which are obtainablefrom the differentiated ES cells of the present invention are useful forstudying the functions of the protein of the present inventioncytologically.

The non-human mammal deficient in expression of the DNA of the presentinvention can be identified from a normal animal by measuring the mRNAamount in the subject animal by a publicly known method, and indirectlycomparing the degrees of expression.

As the non-human mammal, the same examples supra apply.

With respect to the non-human mammal deficient in expression of the DNAof the present invention, the DNA of the present invention can be madeknockout by transfecting a targeting vector, prepared as describedabove, to non-human mammal embryonic stem cells or oocytes thereof, andconducting homologous recombination in which a targeting vector DNAsequence, wherein the DNA of the present invention is inactivated by thetransfection, is replaced with the DNA of the present invention on achromosome of a non-human mammal embryonic stem cell or embryo thereof.

The knockout cells with the DNA of the present invention disrupted canbe identified by Southern hybridization analysis with a DNA fragment onor near the DNA of the present invention as a probe, or by PCR analysisusing a DNA sequence on the targeting vector and another DNA sequencewhich is not included in the targeting vector as primers. When non-humanmammalian embryonic stem cells are used, a cell line wherein the DNA ofthe present invention is inactivated by homologous recombination iscloned; the resulting cloned cell line is injected to, e.g., a non-humanmammalian embryo or blastocyte, at an appropriate stage such as the8-cell stage. The resulting chimeric embryos are transplanted to theuterus of the pseudopregnant non-human mammal. The resulting animal is achimeric animal composed of both cells having the normal locus of theDNA of the present invention and those having an artificially mutatedlocus of the DNA of the present invention.

When some germ cells of the chimeric animal have a mutated locus of theDNA of the present invention, an individual, which entire tissue iscomposed of cells having a mutated locus of the DNA of the presentinvention can be selected from a series of offspring obtained bycrossing between such a chimeric animal and a normal animal, e.g., bycoat color identification, etc. The individuals thus obtained arenormally deficient in heterozygous expression of the protein of thepresent invention. The individuals deficient in homozygous expression ofthe protein of the present invention can be obtained from offspring ofthe intercross between the heterozygotes.

When an oocyte or egg cell is used, a DNA solution may be injected,e.g., to the prenucleus by microinjection thereby to obtain a transgenicnon-human mammal having a targeting vector introduced in a chromosomethereof. From such transgenic non-human mammals, those having a mutationat the locus of the DNA of the present invention can be obtained byselection based on homologous recombination.

As described above, individuals, in which the DNA of the presentinvention is rendered knockout, permit passage rearing under ordinaryrearing conditions, after the individuals obtained by their crossinghave proven to have been knockout.

Furthermore, the genital system may be obtained and maintained byconventional methods. That is, by crossing male and female animals eachhaving the inactivated DNA, homozygote animals having the inactivatedDNA in both loci can be obtained. The homozygotes thus obtained may bereared so that one normal animal and two or more homozygotes areproduced from a mother animal to efficiently obtain such homozygotes. Bycrossing male and female heterozygotes, homozygotes and heterozygoteshaving the inactivated DNA are proliferated and passaged.

The non-human mammal embryonic stem cell in which the DNA of the presentinvention is inactivated is very useful for preparing a non-human mammaldeficient in expression of the DNA of the present invention.

Since the non-human mammal, in which the DNA of the present invention isinactivated, lacks various biological activities derived from theprotein of the present invention, such an animal can be a disease modelsuspected of inactivated biological activities of the protein of thepresent invention and thus, offers an effective study to investigatecauses for and therapy for these diseases.

(10) Method for Screening of a Compound Having theTherapeutic/Prophylactic Effects for Diseases Caused by Deficiency,Damages, etc. of the DNA of the Present Invention

The non-human mammal deficient in expression of the DNA of the presentinvention can be employed for screening of a compound having thetherapeutic/prophylactic effects for diseases (e.g., infectiousdiseases, etc.) caused by deficiency, damages, etc. of the DNA of thepresent invention.

That is, the present invention provides a method for screening of acompound having the therapeutic/prophylactic effects for diseases causedby deficiency, damages, etc. of the DNA of the present invention, whichcomprises administering a test compound to the non-human mammaldeficient in expression of the DNA of the present invention andobserving and measuring a change occurred in the animal.

As the non-human mammal deficient in expression of the DNA of thepresent invention, which can be employed for the screening method, thesame examples as given hereinabove apply.

Examples of the test compounds include peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,vegetable extracts, animal tissue extracts, blood plasma, and the likeand these compounds may be novel compounds or publicly known compounds.

Specifically, the non-human mammal deficient in expression of the DNA ofthe present invention is treated with a test compound, comparison ismade with an intact animal for control and a change in each organ,tissue, disease conditions, etc. of the animal is used as an index toassess the therapeutic/prophylactic effects of the test compound.

For treating an animal to be test with a test compound, for example,oral administration, intravenous injection, etc. are applied and thetreatment is appropriately selected depending upon conditions of thetest animal, properties of the test compound, etc. Furthermore, anamount of a test compound to be administered can be selected dependingon the route for administration, nature of the test compound, and thelike.

For example, when a compound having the therapeutic/prophylactic effectsagainst bronchial asthma is screened, the non-human mammal deficient inexpression of the DNA of the present invention is subjected toimmunization with an antigen (e.g., OVA) followed by inhalation of thesame antigen (e.g., OVA) for airway hyperresponsiveness, the testcompound is administered to the animal and, airway resistance,eosinophil infiltration, etc. of the animal is measured with passage oftime.

In the screening method supra, when a test compound is administered toan animal to be tested and found to inhibit an increase in the airwayresistance of the test animal by the antigen inhalation by at leastabout 10%, preferably at least about 30% and more preferably at leastabout 50%, the test compound can be screened to be a compound having atherapeutic and prophylactic effect for bronchial asthma.

The compound obtainable using the above screening method is a compoundscreened from the test compounds described above and exhibits thetherapeutic and prophylactic effect for diseases (e.g., bronchialasthma, etc.) caused by an increased expression, etc. of the protein ofthe present invention. Therefore, the compound can be employed as a safeand low toxic drug for the treatment/prevention of these diseases.Furthermore, compounds derived from such a compound obtainable by thescreening supra can be similarly employed.

The compound obtained by the screening above may be used in the form ofsalts with physiologically acceptable acids (e.g., inorganic acids ororganic acids) or bases (e.g., alkali metal salts), preferably in theform of physiologically acceptable acid addition salts. Examples of suchsalts are salts with inorganic acids (e.g., hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid), salts with organicacids (e.g., acetic acid, formic acid, propionic acid, fumaric acid,maleic acid, succinic acid, tartaric acid, citric acid, malic acid,oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid)and the like.

A pharmaceutical comprising the compound obtained by the above screeningmethod or salts thereof may be manufactured in a manner similar to themethod for preparing the pharmaceutical comprising the protein of thepresent invention described here in above. Since the pharmaceuticalcomposition thus obtained is safe and low toxic, it can be administeredto human and another mammal (e.g., rat, mouse, guinea pig, rabbit,sheep, swine, bovine, horse, cat, dog, monkey, etc.).

Although the dose of the compound or its salt to be administered variesdepending upon target disease, subject to be administered, route ofadministration, etc., in general, for oral administration to an adult(as 60 kg body weight) for the treatment of, e.g., bronchial asthma, thecompound is administered in a daily dose of about 0.1 to about 100 mg,preferably about 1.0 to about 50 mg, more preferably about 1.0 to about20 mg. For parenteral administration to an adult (as 60 kg body weight)for the treatment of, e.g., bronchial asthma, it is advantageous toadminister the composition intravenously in the form of an injectablepreparation in a single dose of about 0.01 to about 30 mg, preferablyabout 0.1 to about 20 mg, more preferably about 0.1 to about 10 mg,though the single dosage varies depending upon particular subject,particular disease, etc. As for other animals, the composition can beadministered in the above dosage with converting it into that for thebody weight of 60 kg.

(11) Method for Screening a Compound Capable of Promoting or Inhibitingthe Activities of a Promoter to the DNA of the Present Invention

The present invention provides a method for screening a compound orsalts thereof capable of promoting or inhibiting the activities of apromoter to the DNA of the present invention, which comprisesadministering a test compound to a non-human mammal deficient inexpression of the DNA of the present invention and detecting expressionof the reporter gene.

In the screening method supra, the non-human mammal deficient inexpression of the DNA of the present invention is employed, in theaforesaid non-human mammal deficient in expression of the DNA of thepresent invention, as an animal in which the DNA of the presentinvention is inactivated by introducing a reporter gene and the reportergene is expressed under control of a promoter to the DNA of the presentinvention.

The same examples of the test compound apply to specific compounds usedfor the screening.

As the reporter gene, the same specific examples apply to this screeningmethod. Preferably employed are β-galactosidase (lacZ), soluble alkalinephosphatase gene, luciferase gene, and the like.

Since a reporter gene is present under control of a promoter to the DNAof the present invention in the non-human mammal deficient in expressionof the DNA of the present invention, wherein the DNA of the presentinvention is substituted with the reporter gene, the activity of thepromoter can be detected by tracing expression of a substance encoded bythe reporter gene.

When a part of the DNA region encoding the protein of the presentinvention is substituted with, e.g., β-galactosidase gene (lacZ) derivedfrom Escherichia coli, β-galactosidase is expressed in a tissue wherethe protein of the present invention should originally be expressed,instead of the protein of the present invention. Thus, the state ofexpression of the protein of the present invention can be readilyobserved in vivo of an animal by staining with a reagent, e.g.,5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) that is asubstrate for β-galactosidase. Specifically, a mouse deficient in theprotein of the present invention, or its tissue section is fixed withglutaraldehyde, etc. After washing with phosphate buffered saline (PBS),the system is reacted with a staining solution containing X-gal at roomtemperature or about 37° C. for approximately 30 minutes to an hour.After the β-galactosidase reaction is terminated by washing the tissuepreparation with 1 mM EDTA/PBS solution, the color formed is observed.Alternatively, mRNA encoding lacZ may be detected in a conventionalmanner.

The compound or salts thereof obtained using the screening method supraare compounds that are screened from the test compounds described aboveand capable of promoting or inhibiting the promoter activity to the DNAof the present invention.

The compound obtained by the screening method above may be used in theform of salts with physiologically acceptable acids (e.g., inorganicacids or organic acids) or bases (e.g., alkali metal salts), preferablyin the form to of physiologically acceptable acid addition salts.Examples of such salts are salts with inorganic acids (e.g.,hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),salts with organic acids (e.g., acetic acid, formic acid, propionicacid, fumaric acid, maleic acid, succinic acid, tartaric acid, citricacid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,benzenesulfonic acid) and the like.

The compounds or salts thereof capable of promoting the promoteractivity to the DNAa of the present invention can promote expression ofthe protein a of the present invention thereby to promote the activitiesof the protein, and are thus useful as safe and low toxic medicamentsfor the treatment/prevention of diseases such as infectious diseases(e.g., HIV infection, HBV infection, HCV infection, tuberculosisinfection, opportunistic infection, etc.), and the like.

On the other hand, the compounds or salts thereof capable of inhibitingthe promoter activity to the DNAa or DNAb of the present invention caninhibit expression of the protein of the present invention thereby toinhibit the activities of the protein, and are thus useful as safe andlow toxic medicaments for diseases such as lung/chest diseasesaccompanied by inflammation of the lung/airways, including bronchialasthma, chronic obstructive pulmonary disease, etc.

Compounds derived from the compounds obtained by the screening above mayalso be used similarly.

The pharmaceutical comprising the compounds or salts thereof obtained bythe screening method may be manufactured similarly to thepharmaceuticals comprising the protein of the present inventiondescribed above.

Since the pharmaceutical composition thus obtained is safe and lowtoxic, it can be administered to human or another mammal (e.g., rat,mouse, guineapig, rabbit, sheep, swine, bovine, horse, cat, dog, monkey,etc.).

The dose of the compound or salts thereof varies depending on targetdisease, subject to be administered, method for administration, etc.;for example, when the compound capable of inhibiting the promoteractivity to the DNA of the present invention is orally administered forthe treatment of, e.g., bronchial asthma, the dose is normally about 0.1to about 100 mg, preferably about 1.0 to about 50 mg, more preferablyabout 1.0 to about 20 mg per day for adult (as 60 kg body weight). Inparenteral administration for the treatment of, e.g., bronchial asthma,the single dose varies depending on subject to be administered, targetdisease, etc. but it is advantageous to administer, for example, thecompound capable of inhibiting the promoter activity to the DNA of thepresent invention intravenously at a daily dose of about 0.01 to about30 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 toabout 10 mg for adult (as 60 kg body weight). For other animal species,the corresponding dose as converted per 60 kg weight can beadministered.

On the other hand, when the compound capable of promoting the promoteractivity to the DNAa of the present invention is orally administered forthe treatment of, e.g., infectious diseases, the dose is normally about0.1 to about 100 mg, preferably about 1.0 to about 50 mg, morepreferably about 1.0 to about 20 mg per day for adult (as 60 kg bodyweight). In parenteral administration for the treatment of, e.g.,infectious diseases, the single dose varies depending on subject to beadministered, target disease, etc. When the compound capable ofinhibiting the promoter activity to the DNA of the present invention isadministered to an adult (as 60 kg body weight) generally in the form ofinjection, it is advantageous to administer the compound intravenouslyat a daily dose of about 0.01 to about 30 mg, preferably about 0.1 toabout 20 mg, more preferably about 0.1 to about 10 mg. For other animalspecies, the corresponding dose as converted per 60 kg weight can beadministered.

As stated above, the non-human mammal deficient in expression of the DNAof the present invention is extremely useful for screening the compoundor its salt capable of promoting or inhibiting the activity of apromoter to the DNA of the present invention and can greatly contributeto the elucidation of causes for various diseases suspected ofdeficiency in expression of the DNA of the present invention and for thedevelopment of prophylactic/therapeutic agent for these diseases.

Furthermore, a so-called transgenic animal (gene transferred animal) canbe prepared by using a DNA containing a promoter region of the proteinof the present invention, ligating genes encoding various proteinsdownstream and injecting the same into oocyte of an animal. It is thenpossible to synthesize the protein therein specifically and study itsactivity in vivo. When an appropriate reporter gene is ligated to thepromoter site above and a cell line that express the gene isestablished, the resulting system can be utilized for exploring a lowmolecular compound having the action of specifically promoting orinhibiting the in vivo productivity of the protein of the presentinvention, per se. Further analysis of the promoter region enables tofind a new cis-element or a transcription factor bound thereto.

In the specifications and drawings, the codes of bases and amino acidsare denoted in accordance with the IUPAC-IUB Commission on BiochemicalNomenclature or by the common codes in the art, examples of which areshown below. For amino acids that may have the optical isomer, L form ispresented unless otherwise indicated.

DNA deoxyribonucleic acid cDNA complementary deoxyribonucleic acid Aadenine T thymine G guanine C cytosine RNA ribonucleic acid mRNAmessenger ribonucleic acid dATP deoxyadenosine triphosphate dTTPdeoxythymidine triphosphate dGTP deoxyguanosine triphosphate dCTPdeoxycytidine triphosphate ATP adenosine triphosphate EDTAethylenediaminetetraacetic acid SDS sodium dodecyl sulfate Gly glycineAla alanine Val valine Leu leucine Ile isoleucine Ser serine Thrthreonine Cys cysteine Met methionine Glu glutamic acid Asp asparticacid Lys lysine Arg arginine His histidine Phe phenylalanine Tyrtyrosine Trp tryptophan Pro proline Asn asparagine Gln glutamine pGlupyroglutamic acid

Substituents, protecting groups, and reagents used in this specificationare presented as the codes below.

Me methyl group Et ethyl group Bu butyl group Ph phenyl group TCthiazolidine-4(R)-carboxamide group Tos p-toluenesulfonyl CHO formyl Bzlbenzyl Cl₂Bzl 2,6-dichlorobenzyl Bom benzyloxymethyl Z benzyloxycarbonylCl-Z 2-chlorobenzyl oxycarbonyl Br-Z 2-bromobenzyl oxycarbonyl Boct-butoxycarbonyl DNP dinitrophenol Trt trityl Bum t-butoxymethyl FmocN-9-fluorenyl methoxycarbonyl HOBt 1-hydroxybenztriazole HOOBt3,4-dihydro-3-hydroxy-4-oxo-1,2,3 - benzotriazine HONB1-hydroxy-5-norbornene-2,3-dicarboxyimide DCCN,N′-dichlorohexylcarbodiimide

The sequence identification numbers in the sequence listing of thespecification indicate the following sequences.

[SEQ ID NO:1]

This shows the amino acid sequence of the (mature) protein of thepresent invention derived from human stomach.

[SEQ ID NO:2]

This shows the amino acid sequence of the signal peptide of theinvention.

[SEQ ID NO:3]

This shows the base sequence of DNA encoding the human stomach-derivedthe (mature) protein of the present invention having the amino acidsequence represented by SEQ ID NO:1.

[SEQ ID NO:4]

This shows the base sequence of DNA encoding the signal peptide of thepresent invention having the amino acid sequence represented by SEQ IDNO:2.

[SEQ ID NO:5]

This shows the amino acid sequence of the precursor protein of the humanstomach-derived protein of the present invention.

[SEQ ID NO:6]

This shows the base sequence of primer PR1 used in EXAMPLE 1.

[SEQ ID NO:7]

This shows the base sequence of primer PR2 used in EXAMPLE 1.

[SEQ ID NO:8]

This shows the base sequence of primer PR3 used in EXAMPLES 1 and 4.

[SEQ ID NO:9]

This shows the base sequence of primer PR4 used in EXAMPLE 1.

[SEQ ID NO:10]

This shows the base sequence of primer PR5 used in EXAMPLE 1.

[SEQ ID NO:11]

This shows the base sequence of primer PR6 used in EXAMPLE 1.

[SEQ ID NO:12]

This shows the base sequence of primer PR7 used in EXAMPLE 1.

[SEQ ID NO:13]

This shows the base sequence of primer PR8 used in EXAMPLE 1.

[SEQ ID NO:14]

This shows the base sequence of cDNA containing ECF-L full-length geneacquired in EXAMPLE 1.

[SEQ ID NO:15]

This shows the base sequence of ECF-L gene probe used in EXAMPLE 2.

[SEQ ID NO:16]

This shows the base sequence of clone hECF-L-2 acquired in EXAMPLE 5.

[SEQ ID NO:17]

This shows the amino acid sequence of the protein encoding the ECF-Lfull-length gene acquired in EXAMPLE 1.

[SEQ ID NO:18]

This shows the amino acid sequence of ECF-L (mature) protein.

EXAMPLES

Hereinafter the present invention will be described in more detail withreference to EXAMPLES but is not deemed to be limited thereto. The genemanipulation procedures using Escherichia coli were performed accordingto the methods described in the Molecular Cloning.

Escherichia coli JM109/pT7-mECFL bearing the plasmid obtained in EXAMPLE1 by cloning mouse ECF-L full-length DNA fragment to pT7 Blue-T Vectorwas on deposit with the Ministry of International Trade and Industry,Agency of Industrial Science and Technology, National Institute ofBioscience and Human Technology (NIBH) at 1-3, Higashi 1 chome,Tsukuba-shi, Ibaraki-ken, Japan (zip code 305-8566), as the AccessionNumber FERM BP-6881 on Sep. 20, 1999 and with Institute forFermentation, Osaka (IFO) at 17-85, Juso honcho 2-chome, Yodogawa-ku,Osaka-shi, Japan (zipcode 532-8686), as the Accession Number IFO 16315on Aug. 24, 1999.

Escherichia coli DH5α/pcDNA-hECFL bearing the plasmid pcDNA-hECFLobtained in EXAMPLE 6 was on deposit with NIBH as the Accession NumberFERM BP-6878 on Sep. 20, 1999 and with IFO as the Accession Number IFO16312 on Aug. 24, 1999.

Example 1

Cloning of ECF-L Gene Showing Increased Expression in Model Mouse withIncreased Airway Hyperresponsiveness

Model mice with increased airway hyperresponsiveness were preparedthrough sensitization by intraperitoneally injecting 400 μl of salinecontaining 200 μl of OVA (ovalbumin) and 2 mg of alum to BALB/c mice(male, 6 weeks old) and then boosting by intraperitoneal injection of 20μg of saline containing 10 μg of OVA and 1 mg of alum to the animal oneweek after, followed by inhalation of 5% OVA solution dissolved in PBSof {fraction (1/2 )}concentration for 25 minutes under unanaesthetisedand spontaneous respiration conditions over 7 consecutive days from oneweek after. The steroid group was prepared by intraperitoneallyinjecting 1 mg/kg of dexamethasone an hour before the OVA inhalation.Aerozollization was effected using a ultrasonic nebulizer (Soniclizer305, ATOM Medical). Accentuation of airway hyperresponsiveness wasdetermined by the Konzett-Rossler method in terms of bronchoconstrictioninduced by acetylcholine (62.5-2000 μg/kg) given 24 hours after thefinal antigen inhalation. Bronchoalveolar lavage fluid (BALF) wasprepared, after death under pentobarbital anesthesia, by inserting atracheal cannula into the animal and washing 3times with 0.5 ml of PBS.Next, a smear specimen was prepared using cytospin (700 rpm, 1 min.).After Diff-Quick staining and microscopic inspection, the proportion ofmacrophages, eosinophils, neutrophils, lymphocytes and other cells wascalculated.

Poly (A)+RNA used as a sample was prepared by extracting the total RNAfrom the lung/bronchi of normal mice, the lung/bronchi of model micewith increased airway hyperresponsiveness and its dexamethasone group,using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.), andthen passing through oligo-dT cellulose column (manufactured byPharmacia). Using 2 μg aliquot each of these poly (A)+RNAs as thestarting material, cDNA fragments (fragments wherein a part of cDNA isamplified by PCR) specifically expressed in the lung/bronchi of modelmice with increased airway hyperresponsiveness were collected bysubtraction using PCR-select cDNA subtraction kit (manufactured byClontech Laboratories, Inc.). The adaptor sequence for the subtractionadded to the resulting PCR fragment at the both ends thereof was removedby digestion with restriction enzyme RsaI to change to the DNA fragmentwith blunt ends. The fragment was then subcloned to pT7 Blue T-Vector(manufactured by Novagen, Inc.). The DNA base sequence of the subclonedcDNA fragment was decoded, and based on the decoded base sequence,homology search was conducted by blast N using public Geneble database.

The result revealed that 10 out of 120 clones checked on all coincidedwith the base sequence encoding a known mouse ECF-L gene (GENEBANKACCESSION NUMBER: D87757). So, cDNA was synthesized from poly(A)+RNA inthe lung of model mice with increased airway hyperresponsiveness, usingcDNA synthesis kit (manufactured by Takara Shuzo Co., Ltd.). This cDNAwas as a template and PCR was carried out using 2 primers of5′-non-translational region (PR1: SEQ ID NO:6) and 3′-non-translationalregion(PR2: SEQ ID NO:7) of the ECF-L gene to acquire the ECF-Lfull-length gene (SEQ ID NO:14). Using Takara EX Taq (manufactured byTakara Shuzo Co., Ltd.), after incubation 98° C. for a minute, thereaction was carried out by repeating 30 cycles in Thermal Cycler GeneAmp PCR System 9700 (manufactured by Perkin-Elmer, Inc.), in which onecycle is set to include 98° C. for 10 seconds, 60° C. for 1 minute andthen 72° C. for 3 minutes, and finally by reacting at 72° C. for 10minutes. The resulting ECF-L full-length DNA fragment was cloned to pT7Blue-T Vector. Using synthetic primers (PR1 to 8: SEQ ID NOs:6-13),cycle sequencing was conducted to confirm the base sequence of theproduct obtained by fluorescent DNA sequencer (ABI PRISM TM377,manufactured by Perkin-Elmer, Inc.).

Example 2

Analysis on Tissue Distribution on the ECF-L Gene in Model Mice withIncreased Airway Hyperresponsiveness

Each organ (lung, heart, liver, kidney, brain, thymus, spleen, smallintestine, large intestine, stomach) was isolated from normal mice andmodel mice with increased airway hyperresponsiveness, and total RNAswere prepared therefrom, using ISOGEN (manufactured by Wako PureChemical Industries, Ltd.). The total RNAs were passed through oligo-dTcellulose column (manufactured by pharmacia, Inc.) to preparepoly(A)+RNAs. After 0.5 μg of this poly(A)+RNAs were electrophoresed on1.1% agarose gel electrophoresis containing 2.2 M formalin, the RNAswere blotted to nylon membrane filters (Hybond-N+, made by AmershamPharmacia Biotech, Inc.) by capillary blotting for 18 hours. The RNAswere fixed on the nylon membrane filters through UV treatment, followedby hybridization at 65° C. in Express Hyb Hybridization Solution(manufactured by Clontech Laboratories, Inc.). On the other hand, one ofthe ECF-L cDNA fragments shown as a probe in EXAMPLE 1 (SEQ ID NO:15)was labeled with [α-³²P]dCTP and Bca BEST Labeling Kit (manufactured byTakara Shuzo Co., Ltd.). Hybridization was carried out at 65° C. for 2hours in Express Hyb Hybridization Solution. Filters were finally rinsedwith 0.1×SSC in 0.1% SDS solution at 50° C. followed by detection usingBAS-2000 (manufactured by Fuji Photo Film Co., Ltd.). As a result,expression of the ECF-L gene (mRNA) was observed in the lung, thymus andstomach in normal mice. In mice with increased airwayhyperresponsiveness, the expression was markedly observed in the lung,indicating that the expression was strongly induced with increasedairway hyperresponsiveness. An increase of expression was also noted inthe thymus and stomach (FIG. 1).

Example 3

Analysis on the ECF-L Gene with Passage of Time in Model Mice withIncreased Airway Hyperresponsiveness

Using the model mice with increased airway hyperresponsiveness explainedin EXAMPLE 1 above, the increased airway hyperresponsiveness and thecount of infiltrated cells into alveolar lavage fluids were measuredbefore OVA inhalation and on Days 2, 3, 4, 5, 6 and 7 after OVAinhalation as in EXAMPLE 1 (FIGS. 2, 3 and 4 ). In addition, the lungbefore OVA inhalation and on Days 1, 2, 3, 5 and 7 after OVA inhalationwas isolated, and subjected to Northern blotting analysis as in EXAMPLE2 (FIG. 5). As a result, the increased airway hyperresponsiveness andinfiltration of eosinophils into alveolar lavage fluids were induced onor after Day 4 after OVA inhalation, whereas expression of the ECF-Lgene was markedly induced from Day 2 after OVA inhalation. That is,expression of the ECF-L gene occurred prior to the increased airwayhyperresponsiveness and eosinophil infiltration, but the ECF-L gene wasnot expressed as the outcome of airway inflammation, suggesting thepossibility that induction of the ECF-L gene expression would cause theincreased airway hyperresponsiveness and eosinophil infiltration intoalveolar lavage fluids.

Example 4

Identification of the ECF-L Gene Expression Site in Model Mice withIncreased Airway Hyperresponsiveness

After perfusion in lung of normal mice and model mice with increasedairway hyperresponsiveness and fixation with 4% paraformaldehyde, thelung was isolated and fixed at 4° C. overnight. Thereafter, asucrose-HBSS solution was replaced to finally reach 18% sucrose-HBSSsolution by gradually increasing the concentration of sucrose, and thelung was frozen in dry ice. After allowing to stand in a cryostat at−14° C. for 3 hours, the frozen lung was cut into a thickness of 10-15μl and put up on an APS-coated slide glass. For preparing a DIG-labeledprobe, the ECF-L DNA fragment of 0.6 kb was amplified by PCR using theECF-L full-length gene fragment obtained in EXAMPLE 1 as a template andusing synthetic primers (PR3: SEQ ID NO:8, PR6: SEQ ID NO:11). UsingTakara EX Taq (manufactured by Takara Shuzo Co., Ltd.), after incubation94° C. for 1 minute, the reaction was carried out by repeating 30 cyclesin Thermal Cycler Gene Amp PCR System 9700 (manufactured byPerkin-Elmer, Inc.), in which one cycle is set to include 94° C. for 10seconds, 60° C. for 30 seconds and then 72° C. for90 seconds, andfinally by reacting at 72° C. for 10 minutes. The amplified DNA fragmentwas inserted into PCRII-TOPO vector (manufactured by Invitrogen, Inc.),and extended from both sides of the vector by SF6 RNA polymerase and T7RNA polymerase using DIG Labeling Kit (manufactured by BoehringerMannheim) in accordance with the manual attached to prepare DIG-labeledantisense and sense probe. In situ hybridization was conducted usingISHR Starter Kit (manufactured by Nippon Gene Co., Ltd.) in accordancewith the manual attached thereto. As a result, it was found that theECF-L gene was highly expressed in the model mice with increased airwayhyperresponsiveness. In the normal mice, no expression of the ECF-L genewas observed in any part of the lung (FIG. 6).

Example 5

Cloning of a Gene Encoding Human-derived ECF-L-like Protein

Using the mouse ECF-L full length gene shown in EXAMPLE 1 as a probe,Northern blotting analysis was performed on human RNA master blot(manufactured by Clontech Laboratories, Inc.). Hybridization was carriedout at 68° C. for 2 hours in Express Hyb Hybridization Solution, andrinsing was finally made with 0.1×SSC in 0.1% SDS solution at 50° C. Fordetection, BAS-2000 (manufactured by Fuji Photo Film Co., Ltd.) wasused. As a result, a marked signal was detected in the stomach. It wasthus decided to acquire human counterpart of the ECF-L gene from humanstomach cDNA library.

After human stomach 5′-stretch plus cDNA library (using λgt11 phage DNAas a vector, manufactured by Clontech Laboratories, Inc.) was infectedto E. coli Y1090r⁻, about 200,000 plaques each was seeded in 7 soft agarplates and cultured overnight at 37° C. to form plaques. After theplaques were transferred to a nylon membrane filter (Hybond-N, made byAmersham Pharmacia Biotech, Inc.), the plaques were treated sequentiallywith a denaturation solution (0.5N NaOH, 1.5M NaCl), a neutralizingsolution (0.5M Tris Cl pH 8.0, 1.5M NaCl) and 2×SSC. After air drying,UV rays were irradiated to fix phage DNA on the nylon membrane filter.Plaque hybridization was carried out at 68° C. for at least 3 hours inExpress Hyb Hybridization Solution containing a labeled probe. After thefilter was rinsed finally with 0.1×SSC in 0.1% SDS solution at 50° C.,autoradiogram was taken to survey plaques hybridizable to the probe.Lambda DNA was prepared from 7 phage clones hECF-L-1, 2, 3, 10, 13, aand b, which were purified to single clones by repeating this procedure,using QIAGEN LAMBDA MINIKIT (manufactured by Qiagen) in accordance withthe manual attached. Subsequently, a reaction was carried out usingBigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured byPerkin-Elmer, Inc.), and the base sequence of the cDNA fragment insertedwas determined using DNA Sequencer 377 (manufactured by Perkin-Elmer,Inc.). The results revealed that the 7 clones acquired contained thesame DNA fragment and clone hECF-L-2 containing the longest DNA fragmenthad a 1678 bases (SEQ ID NO:16). The cDNA fragment encoded ahuman-derived new ECF-L-like protein consisting of 476 amino acids (SEQID NO:5). The protein had 70% homology in its base level and 68%homology in its amino acid level, to mouse ECF-L (FIGS. 7 and 8).Further homology search by blast N using the Geneble database revealedthat the cDNA was a novel gene belonging to a chitinase (FIGS. 9 and10). This protein has a sequence reserved at the catalytic center ofchitinase, and showed 57% homology in the DNA level and 51% homology inthe amino acid level, to human chitotrioxidase [J. Biol. Chem., 270,26252 (1995)], which is reported to be the only one chitinase in human.

Example 6

Construction of Vector to Express a Gene Encoding Human-derived NewECF-L-like Protein in Animal Cells

After Agtll phage DNA, in which the gene encoding human-derivedECF-L-like protein shown in EXAMPLE 5had been inserted, was digestedwith EcoRI, the resulting DNA fragment of 1.7 kbp containing the geneencoding human-derived ECF-L-like protein was inserted into pcDNA 3.1plasmid (manufactured by Invitrogen, Inc.) likewise digested with EcoRIto acquire plasmid pcDNA-hECFL bearing the gene encoding human-derivedECF-L-like protein downstream cytomegalovirus enhancer/promoter andhaving a neomycin resistant gene as a selection marker.

Example 7

Expression of the Gene Encoding Human-derived Novel ECF-L-like Proteinin COS-7 Cells and Assay for Chitinase Activity

COS-7 cells, 9×10⁵, were cultured for 24 hours in Dulbecco's ModifiedEagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), and7.5 μg of the expression plasmid (pcDNA-hECFL) shown in EXAMPLE 6 wastransfected using lipofectamine (GIBCO BRL). Two days after thetransfection, the medium was replaced with FCS-free DMEM and incubationwas continued for 4 days to obtain the culture supernatant. Thechitinase activity was assayed according to the report by Renkema, G. H.et al. [J. Biol. Chem., 20, 2198 (1995)]. That is, 100 μl of the culturesupernatant described above was added to 100 μl of a reaction buffer(McIlvain buffer, pH 5.2), in which a fluorescent substrate(4-methylumbelliferyl β-D-N,N′-diacetylchitobioside (4MU-chitobioside),4-methylumbellieryl β-D-N,N,N′-triacetylchitotrioside(4-MU-chitotrioside)) was dissolved in a final concentration of 0.027mM, followed by incubation at 37° C. for 30 minutes. By adding 1 ml of areaction termination buffer (0.3M glycine/NaOH buffer, pH10.6), thereaction was terminated, and chitinase activity was measured using afluorescence measurement device (excited wavelength of 355 nm,measurement wavelength of 460 nm). For negative control, the culturesupernatant from plasmid-non-transfected COS-7 cells was used and, 0.001U of Serratia marcescens chitinase was used for positive control. As aresult, the chitinase activity was detected in the culture supernatantfrom the expression plasmid (pcDNA-hECFL)-transfected COS-7 cells.

Example 8

Analysis on Tissue Distribution of the Gene Encoding Human-derived NovelECF-L-like Protein

Using the DNA fragment (1.7 kbp) inserted with the gene encodinghuman-derived novel ECF-L-like protein shown in EXAMPLE 1 as a probe,Northern blotting analysis was performed on human RNA master blot(manufactured by clontech Laboratories, Inc.). Hybridization was carriedout at 68° C. for 2 hours in Express Hyb Hybridization Solutioncontaining a labeled probe, and rinsing was made finally with 0.1×SSC in0.1% SDS solution at 50° C. For detection, BAS-2000 (manufactured byFuji Photo Film Co., Ltd.) was used. As a result, a marked signal wasdetected in the stomach and the expression was observed also in the lungand embryonic lung (FIG. 11).

INDUSTRIAL APPLICABILITY

The protein of the present invention and the DNA encoding the same canbe employed as therapeutic/prophylactic agents for diseases such asinfectious diseases. The protein of the present invention is also usefulas a reagent for screening a compound or its salts capable of promotingor inhibiting the activities of the protein of the present invention.Furthermore, a compound or its salts capable of inhibiting theactivities of the protein of the present invention, or a neutralizationantibody that inhibits the activities of the protein of the presentinvention can be used as therapeutic/prophylactic agents for diseasesincluding bronchial asthma, chronic obstructive pulmonary disease, etc.Moreover, the antibodies against the protein of the present inventioncan recognize the protein of the present invention specifically and canbe used for quantification of the protein of the present invention in atest sample fluid.

1. An isolated protein having the amino acid sequence represented by SEQID NO: 1, or a salt thereof.
 2. An isolated signal peptide having theamino acid sequence represented by SEQ ID NO: 2, or a salt thereof. 3.An isolated polynucleotide comprising a DNA sequence encoding theprotein according to claim 1 and having the base sequence represented bySEQ ID NO:3.
 4. An isolated polynucleotide comprising a DNA sequenceencoding the signal peptide according to claim 1 and having the basesequence represented by SEQ ID NO:4.
 5. A recombinant vector comprisingthe DNA sequence according to claim
 3. 6. A transformant transformedwith the recombinant vector according to claim
 5. 7. A method ofmanufacturing the protein according to claim 1 or a salt thereof, whichcomprises culturing a transformed host cell comprising an expressibleDNA expression vector encoding for said protein, under conditionssuitable for the expression of said protein for sufficient time toexpress said protein, and collecting the same.
 8. A pharmaceuticalcomprising the protein according to claim 1 or a salt thereof, and apharmaceutically acceptable carrier, diluent or excipient.
 9. Apharmaceutical comprising the DNA according to claim 3, and apharmaceutically acceptable carrier, excipient or diluent.